Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators

Abstract

In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA-(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA-(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA-(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.

Figure 1.

CcpA–(HPr-Ser46-P)–cre site promiscuity. (A) Sequences of the three cre sites, syn, ackA2 and gntR-down, used in the study. Asterisks over the sequences show the locations where the sites differ. Below the three sites used in the study are eight additional cre sites. These sequences are shown to illustrate the natural variation of in vivo cre sites (36,50–54). (B) Fluorescence polarization-based binding isotherms of CcpA–(HPr-Ser46-P) syn cre, ackA2 cre and gntR-down cre (from left to right). The K_d calculated for each experiment is indicated in each isotherm.

Figure 2.

SPR analysis of CcpA–(HPr-Ser46-P) binding to cre elements reveals similar association and dissociation rates. The titrations were carried out with 5 or 20 to 100 nM CcpA. To reduce bulk effects, the running buffer was supplemented with 75 µM HPr-Ser46-P in the gntR-down cre experiment (A) and with 50 µM HPr-Ser46-P in the ackA2 and syn cre experiments (B and C). Under these conditions, CcpA is completely bound by HPr-Ser46-P (39). Dashed lines represent the measured data and bold lines are the best calculated fits for the association reactions.

Figure 3.

Structures of CcpA–(HPr-Ser46-P) bound to three cre sites. Shown from left to right are the ribbon diagrams of the CcpA–(HPr-Ser46-P)–syn, CcpA–(HPr-Ser46-P)–ackA2 and CcpA–(HPr-Ser46-P)–gntR-down structures. The CcpA subunits in the dimer are coloured cyan, the HPr-Ser46-P molecules are coloured red. The α-helices are shown as coils and β-strands as arrows. The DNA is shown as sticks with phosphorus, nitrogen, oxygen and carbon atoms coloured magenta, blue, red and yellow, respectively. Each DNA phosphate backbone is depicted as a magenta tube. The Ser46-P residues are shown as solid surface representations. This figure, Figures 4A–C, 5A and B and 6 were made with PyMOL (55).

Figure 4.

CcpA–(HPr-Ser46-P)–cre binding requires protein and DNA flexibility. Different orientations of the HTHLH modules and the DNA conformations of CcpA–(HPr-Ser46-P) bound cre sites. (A) View of the DNA-binding HTHLH modules and cognate DNA sites after superimposition of the hinge helix and C-domain of CcpA of each structure. Note that the positions of the HTH motifs are significantly different with the greatest change seen for the HTH motif of the CcpA bound to the syn cre (yellow). The structures of the HTHLH, hinge helices and DNA backbones of the CcpA–(HPr-Ser46-P)–ackA2 and CcpA–(HPr-Ser46-P)–gntR-down complexes are coloured blue and red, respectively. (B) View of the superimposed DNA phosphate backbones of the cre sites, coloured as in Figure 4A and rotated ∼45° to allow a view into the enlarged DNA minor grooves. (C) View of the overlaid cre DNAs with all atoms included. Coloured as in Figure 4A.

Figure 5.

CcpA–cre interactions. (A) Close up view of the Arg22 interaction with Gua3 and the van der Waals contact between the side chain Cβ of Asn29 with Thy2 (indicated by double headed arrow). Also note the stacking of the Arg22 side chain with the unstacked Thy2 nucleobase. (B) Close up of the hinge helix interactions with the rolled CpG step (for clarity, only one of the two hinge helices is shown). Weak hydrogen bonds are observed between the carbonyl oxygen moiety of residue Ala53 to the N2 atom of Gua9 and the amide nitrogen of Ala57 and the O2 of Cyt8. The Leu56 side chains (highlighted as transparent surfaces) fit optimally into the widened minor groove. Figures 5A and B show contacts from the CcpA–(HPr-Ser46-P)–ackA2 structure. The same interactions are observed in the CcpA–(HPr-Ser46-P)–gntR-down and CcpA–(HPr-Ser46-P)–syn structures. (C) From left to right are schematic representations of the interactions between CcpA and the syn, ackA2 and gntR-down sites. The strands are labelled 1A–16A on one strand and 1B–16B on the other strand. Bases are represented as rectangles and labelled according to sequence. The ribose groups are shown as pentagons. van der Waals contacts between side chains and bases are indicated by black diamonds and hydrogen bonds, by black arrows. Hydrogen bonds to the backbone phosphate groups are shown as coloured arrows. Peptide backbone–DNA hydrogen bonds are designated further with the word ‘amide’ with the exception of the A53 and L56 DNA hydrogen bonds.

Figure 6.

Key properties that allow CcpA to function as a global regulator. The DNA-binding properties of CcpA that are key for its global function include the utilization of two separable DNA-binding modules, the HTHLH motifs, which contacts the major groove, and the hinge helices, which contact the minor groove. The flexible linkage between the modules and an abundance of non-polar residues residing on the ‘recognition helix’ (α2), allow multiple, relatively non-specific nucleobase contacts and permit plasticity in both binding conformation and nucleobase recognition. The HTHLH (module 1) is shown as a pink ribbon whilst the hinge helix (module 2) is shown as a yellow ribbon. The flexible attachment of the HTHLH is indicated by an arrow and the extent of the flexibility is indicated by the overlays of the syn bound and the ackA2 bound structures, which show the largest deviations. The gntR–down complex has a HTHLH position that is in between these two extremes. Residues that are involved in the recognition of the conserved G–C base pair (residue R22) and the central CpG step (residue L56) are shown as solid blue spheres. The residues of the recognition helix that make van der Waals contacts to the cre sites and allow DNA sequence promiscuity are shown as dotted cyan surfaces. The DNA sugar phosphate backbone is shown as a grey tube.