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Originally published In Press as doi:10.1074/jbc.M509977200 on November 29, 2005

J. Biol. Chem., Vol. 281, Issue 10, 6793-6800, March 10, 2006
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Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation*

Maria A. Schumacher{ddagger}, Gerald Seidel§, Wolfgang Hillen§, and Richard G. Brennan{ddagger}1

From the {ddagger}Department of Biochemistry & Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 and the §Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universität Erlangen-Nürnberg, Staudstr. 5, 91058 Erlangen, Germany

Received for publication, September 12, 2005 , and in revised form, November 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser46-P or Crh-Ser46-P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser46-P more weakly than HPr-Ser46-P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser46-P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser46-P)-DNA complex. The structure reveals that Crh-Ser46-P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser46-P. The reduced affinity of Crh-Ser46-P for CcpA as compared with that of HPr-Ser46 P is explained by weaker Crh-Ser46-P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix {alpha} 2 of the second contact region (contact region II) of Crh-Ser46-P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Carbon catabolite repression/regulation (CCR)2 is a global regulatory mechanism utilized by bacteria to select, out of a mixture of compounds, the carbon source providing the optimal growth advantage (1-3). CCR is mediated largely at the level of transcription. The master transcriptional regulator of CCR in bacilli and other Gram-positive bacteria with low GC content is the catabolite control protein A (CcpA) (4-10). CcpA binds to catabolite responsive elements (cre) to mediate its effect (11, 12). Approximately 10% of the Bacillus subtilis genome is under regulation by CcpA, underscoring its vital metabolic role (13).

CcpA is a member of the LacI-GalR family of transcription regulators (14). LacI-GalR proteins contain a 60-residue N-terminal DNA binding domain that connects to a larger C-terminal domain. The C-terminal domain consists of N- and C-subdomains connected by a hinge region. Movements about the hinge lead to "open" and "closed" states of the domain. For most LacI-GalR members, small molecule ligands act as either inducers or corepressors by binding in the cavity between the subdomains to stabilize the open or closed form (15-20). Although some studies suggest that glucose 6-phosphate and fructose 1,6-bisphosphate act as corepressors of CcpA, these data are not unequivocal (21-23). By contrast, multiple studies have demonstrated that the Ser46-phosphorylated form of HPr binds CcpA as a corepressor, activating CcpA to bind cre sites (6, 24-26). The recent structure of the CcpA-(HPr-Ser46-P)-cre revealed the mechanism by which HPr-Ser46-P functions as a corepressor for CcpA (27). Strikingly, this DNA binding activation mechanism is different from those of PurR and LacI in that CcpA utilizes a two-component phosphoprotein that is induced and stabilized by closure of its N- and C-subdomains. This mechanism involves both a rotation of CcpA subdomains as well as a relocation of the key residue Thr61, which is located at the interface of the DNA-binding and corepressor-binding domains. The repositioning of this residue leads to a juxtaposition of the DNA-binding domains to permit hinge helix formation in the presence of cognate DNA (15, 16).

In addition to HPr, a structural and functional homologue, Crh (for catabolite repression HPr) has been identified, but only in bacilli (28). Crh shows 45% sequence identity with HPr but lacks the His15 residue found in HPr that is phosphorylated to function in a phosphoenolpyruvate:sugar phosphotransferase system. Crh, instead contains a conservative substitution to glutamine at this position (29, 30). However, like HPr, Crh does contain the Ser46 residue that is phosphorylated by the enzyme HPr kinase/phosphorylase, and Crh has been demonstrated to function in CCR (28, 31). Interestingly, Crh-Ser46-P can only partially substitute for HPr-Ser46-P in CCR, whereas HPr-Ser46-P can completely substitute for Crh-Ser46-P in this pathway (28, 32). These findings appear to be explained by the finding that HPr is produced much more efficiently than Crh under strong conditions of CCR, resulting in up to 100-fold more HPr than Crh (33). Indeed, when bacteria utilize succinate or citrate as their major carbon source, the difference in HPr and Crh levels is only ~10-fold and under these conditions citM, a gene encoding the Mg2+-citrate transporter, is specifically repressed by Crh but not by HPr (33).

HPr and Crh have highly homologous sequences and are structurally similar, indicating that the phosphorylated forms of these proteins likely bind CcpA similarly and elicit the same allosteric DNA binding switch. Examination of the CcpA-(HPr-Ser46-P)-cre complex structure shows that, with the exception of two residues (HPr -> Crh: H15Q, T20A), the expected CcpA binding interfaces of the two proteins should be identical. Modeling studies using the CcpA-(HPr-Ser46-P)-cre structure suggested that Crh residue Gln15 would be able to interact with CcpA residue Asp 296 in a manner similar to the interaction observed between His15 and Asp296 in the CcpA-(HPr-Ser46-P)-cre structure (27). Interestingly, biochemical studies have revealed that Crh-Ser46-P binds CcpA with up to 10-fold reduction in affinity as compared with HPr-Ser46-P (34), thereby indicating differences in their binding mechanisms.


Figure 1
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  		FIGURE 1.
Structure of the CcpA-(Crh-Ser46-P)- cre complex. A, ribbon diagram of the CcpA-(Crh-Ser46-P)-cre complex. The CcpA subunits are colored blue and cyan. The Crh-Ser46-P subunits are colored red, and the DNA is shown as sticks with oxygen, nitrogen, carbon, and phosphorus atoms colored red, dark blue, light blue, and magenta, respectively. Also shown as sticks are CcpA residues Arg303, Lys307, and Arg324 and Crh-Ser46-P residue Gln15. Crh residues Ser46-P are shown as red sticks. This figure and Figs. 2B, 3, B and C, 4, A and B, and 5 were made with SwissPdb Viewer (46) and rendered with POVRAY (POV-Ray, Persistence of Vision Raytracer, version 3.1). B, simulated annealing composite omit electron density map of the CcpA Arg324-(Crh-Ser46-P) Gln15 contact region. The electron density is shown as a blue mesh and contoured at 1.5 {sigma}. Labeled are CcpA residues Arg324 and Asp296 and Crh-Ser46-P residues Gln15 and Ala16. Carbon, nitrogen, and oxygen atoms are colored yellow, blue, and red, respectively. Hydrogen bonds are represented as white dashed lines. This figure was made with O (38).

 
Previous structural studies on Crh reveal that it has essentially the same fold as HPr. However, a recent crystal structure of Crh showed a domain-swapped dimer and NMR studies have provided evidence that, at high concentrations, Crh can form a mixture of monomers and dimers (35, 36). From these studies a dimer-dimer type of interaction between Crh and CcpA was suggested. By contrast, the CcpA-(HPr-Ser46-P)-cre structure, which showed two monomers of HPr-Ser46-P bound per CcpA dimer, indicates that dimerization is not likely relevant in the binding of Ser46-phosphorylated, HPr-like corepressors to CcpA (27). Moreover, recent biochemical studies, which examined the interaction of CcpA with HPr-Ser46-P and Crh-Ser46-P, indicate that both proteins bind CcpA as a monomer (34). The possible relevance of a Crh-Ser46-P dimerization in its binding to CcpA remains unclear and whether dimerization or other structural alterations of Crh explains the reduced binding affinity of Crh-Ser46-P for CcpA is not known. Therefore, to determine the molecular basis for Crh-Ser46-P binding to CcpA and thus, gain insight into its reduced affinity for CcpA as compared with HPr-Ser46-P, we determined the crystal structure of a CcpA-(Crh-Ser46-P)-cre complex to 2.96-Å resolution.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Preparation, Crystallization, and Data Collection—Bacillus subtilis and Bacillus megaterium CcpA and B. subtilis Crh-Ser46-P proteins were overexpressed and purified as described (27, 34). Both B. subtilis and B. megaterium His-tagged CcpA were used in crystallization screens as sequence alignment of CcpA proteins revealed that the HPr-Ser46-P interacting surfaces of these proteins are identical (27). The B. megaterium and B. subtilis Crh proteins (both 88 residues) share 64% sequence identity but their predicted CcpA interacting surfaces share 100% sequence identity3. Data quality crystals were obtained using only B. megaterium CcpA, B. subtilis Crh-Ser46-P, and the 16-bp cre duplex with the sequence of one strand 5'-CTGTTAGCGCTTTCAG-3'. Crystals were grown at 298 K using the hanging drop vapor diffusion method by mixing the stoichiometric CcpA(dimer)-(Crh-Ser46-P)(2 monomers)-cre duplex complex 1:1 with a reservoir solution of 22% PEG MME 3350, 0.2 M sodium iodide and sealing the drop over 1 ml of the reservoir. The crystals are monoclinic, space group C2, with a = 83.69 Å, b = 158.10 Å, c = 125.47 Å, and beta = 100.73°. For cryoprotection, glycerol was added to a final concentration of 35%. X-ray intensity data were collected at the Advanced Light Source beamline 8.2.1 at 100 K, processed with MOSFLM, and scaled with SCALA (Table 1).


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  		TABLE 1
Selected crystallographic statistics of the CcpA-(Crh-Ser46-P)-cre complex

 
Structure Determination—The CcpA-(CrH-Ser46-P)-cre structure was determined by molecular replacement using the CcpA-(HPr-Ser46-P)-cre structure, with the solvent removed (27), as a search model and the program EPMR (37). Searching with a single subunit of CcpA, its bound HPr-Ser46-P corepressor, and a cre half-site produced three clear solutions: two of which formed complex CcpA-(CrH-Ser46-P)-cre dimer, and the third, a monomer, that when the crystallographic symmetry was applied produced a dimer. This starting model was first subjected to rigid body refinement, in which each CcpA subunit, Crh-Ser46-P molecule and DNA half-site, were treated as rigid units (38, 39). This was followed by multiple cycles of simulated annealing and positional/thermal parameter refinement in CNS and rebuilding in O (38, 39).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Structure of the CcpA-(Crh-Ser46-P)-cre Complex—The CcpA-Crh-Ser46-P-cre DNA complex was crystallized using equimolar amounts of CcpA monomer, Crh-Ser46-P (monomer), and the 16-bp cre site (with one strand of the sequence 5'-CTGTTAGCGCTTTCAG-3'). This sequence was also used in the determination of the CcpA-(HPr-Ser46-P)-cre structure to allow comparison of the CcpA-DNA contacts when bound by the different CcpA-phosphoprotein corepressor complexes (27). The structure was solved by molecular replacement using the CcpA-(HPr-Ser46-P)-cre structure as a search model ("Materials and Methods," Fig. 1, A and B, and Table 1). The crystallographic asymmetric unit contains one dimeric CcpA-(Crh-Ser46-P)-cre complex and one monomeric complex in which crystallographic symmetry generates the dimer. Thus, the structure provides three independent views of CcpA-(Crh-Ser46-P) and CcpA-cre interactions. The final model includes residues 1-42, 46-332 of one CcpA subunit and residues 1-332 of the other two CcpA subunits, residues 2-84 of the three Crh-Ser46-P subunits, all nucleotides of the three 16-bp cre strands, 11 iodide atoms, and 46 solvent molecules. The Rwork and Rfree are 23.2 and 29.8%, respectively, to 2.96-Å resolution and selected model statistics (40) are given in Table 1.

The structure of CcpA, like LacI-GalR members PurR and LacI, consists of a DNA-binding domain (residues 1-59) and a dimerization/corepressor (Crh-Ser46-P)-binding domain (residues 60-332) (15-19). The DNA-binding domain can be divided into two regions, {alpha}1 through {alpha}3, which form the HTH containing three-helix bundle, and {alpha}4, which forms the minor groove binding hinge helix (Fig. 1A). As is characteristic of the LacI-GalR proteins, there is a dramatic kink in the conserved central CpG step of the cre DNA, which is induced by the two conserved symmetry related leucines (Leu56) located on the hinge helices. The conformation of CcpA in the CcpA-(Crh-Ser46-P)-cre structure is essentially identical to that found in the CcpA-(HPr-Ser46-P)-cre structure; superimpositions of the C{alpha} atoms of each CcpA subunit from the CcpA-(Crh-Ser46-P)-cre structure onto a CcpA subunit from the CcpA-(HPr-Ser46-P)-cre structure results in root mean square deviations (r.m.s. deviations) of ~0.70 Å. Similar overlays of the CcpA dimers from each structure result in r.m.s. deviations of ~1.0 Å for all C{alpha} atoms. The larger deviation in superimposition of the dimer reflects the flexible attachment between helix {alpha} 3 and the hinge helix, {alpha}4 (see below).

The CcpA-(Crh-Ser46-P)-cre structure reveals that each Crh-Ser46-P binds the surface of CcpA with a stoichiometry of one Crh-Ser46-P molecule per CcpA subunit. As anticipated, the CcpA-(Crh-Ser46-P) binding interface is similar to the CcpA-(HPr-Ser46-P) interface, but not identical (Fig. 1A) (6, 27). Crh-Ser46-P binds CcpA as a monomer in all three crystallographically independent complexes, clearly demonstrating that a Crh dimer is not utilized in formation of the CcpA-(Crh-Ser46-P)-cre ternary complex. Moreover, modeling of the domain-swapped Crh dimer into the complex reveals that steric clash between the N-subdomain of CcpA and the second subunit of Crh (data not shown) would preclude an identical binding mode.

Comparison of the CcpA-bound HPr-Ser46-P and Crh-Ser46-P Molecules—The three crystallographically independent Crh-Ser46-P proteins bound to CcpA have the same structures as evidenced from the average r.m.s. deviation of 0.51 Å for the pairwise superimpositions of all 84 Crh C{alpha} atoms. Crh-Ser46-P contains three {alpha}-helices ({alpha}1, residues 18-27; {alpha}2, residues 47-52; {alpha}3, residues 70-81) and four beta-strands (beta1, residues 2-6; beta2, residues 32-37; beta3, residues 41-44; beta4, residues 61-65) (Fig. 1). Comparison of the Crh-Ser46-P structure in our ternary complex to that of HPr-Ser46-P in the CcpA-(HPr-Ser46-P)-cre structure results in a r.m.s. deviation of 1.2 Å for 80 corresponding C{alpha} atoms, showing that Crh-Ser46-P and HPr-Ser46-P adopt essentially the same structure when bound to CcpA. Only two significant differences are found between the HPr-Ser46-P and Crh-Ser46-P structures bound to CcpA. The first is the conformation of helix {alpha}3, which is tilted slightly differently in the two proteins and the second is the absence of HPr-Ser46-P beta strand beta5 (residues 86-88) from Crh-Ser46-P. These secondary elements are located distal to the regions that interact with CcpA and likely have little impact on Crh-Ser46-P and HPr-Ser46-P binding to CcpA.

Two structures of Crh, but not Crh-Ser46-P, have been reported; one determined by NMR and the other by crystallography. The NMR structure shows a monomer with the same overall fold as Crh in our structure (36). However, the NMR analysis suggested that Crh dimerizes at high concentrations and a subsequent crystal structure revealed a domain-swapped dimer in which the N-terminal beta strand, beta1, exchanges subunits (35). Superimposition of the C{alpha} atoms of our Crh-Ser46-P structure onto the corresponding C{alpha} atoms of a Crh subunit in the domain-swapped structure, excluding residues 1-12, which are involved in domain exchange, results in r.m.s. deviation of 1.1 Å. Intriguingly, the crystal structure of Crh-Ser46-P is also identically domain swapped. 4What role, if any, Crh dimerization may play in vivo is unclear. However, our structure, obtained under high protein concentrations (200 µM) clearly reveals that the monomer of Crh-Ser46-P functions as a corepressor for CcpA, a finding consistent with recent studies examining CcpA binding to Crh-Ser46-P and HPr-Ser-46-P by surface plasmon resonance (34).

Flexible DNA Binding by the CcpA HTH Elements—The CcpA-(Crh-Ser46-P)-cre structure provides three crystallographically independent views of the interaction between CcpA and the 16-bp cre. CcpA, like other LacI-GalR proteins, kinks its DNA binding site to allow formation of operator-specific, HTH-major groove contacts (41, 42). As expected, these contacts are essentially identical to those observed in the CcpA-(HPr-Ser46-P)-cre structure, in which the same oligodeoxynucleotide was used in crystallization (27) (Fig. 2A). The global DNA bend angle, induced by partial interaction of the dyad-related hinge helix residues Leu55, the "leucine levers," 31°, is the smallest bend angle observed thus far for a LacI-GalR protein; the bend angles of the DNA bound by PurR and LacI are ~50 and ~40°, respectively, whereas the global bend angle observed in the CcpA-(HPr-Ser46-P)-cre structure, which, again, is the same DNA site as in the CcpA-(Crh-Ser46-P)-cre structure, is 35° (15-19, 27).

The smaller DNA bend angle observed in the CcpA-(Crh-Ser46-P)-cre structure as compared with the other LacI proteins appears to be the result of the different docking modes of their HTH motifs onto the major grooves rather than any differences in the partial intercalation of the hinge helices. Superimpositions of the dimer/corepressor binding domains reveal that the positions of the hinge helices (as well as the DNA around the central CpG step) are highly conserved structurally (Fig. 2B). By contrast, the three-helix bundle, composed of the HTH motif and {alpha}3, rotate as a unit about a flexible loop that links helices {alpha}3 and {alpha}4. Such flexibility permits the CcpA HTH motifs to adjust individually to accommodate to the precise major groove sequence of the DNA as well as the conformation of the DNA, the latter of which may be influenced by the adjacent DNA or adjacently bound proteins. Similar plasticity has been observed in structures of the Lac repressor (41, 42). However, there appears to be a thermodynamic cost associated with such binding flexibility as CcpA binds the amyE cre nearly 12-fold more tightly than the xyl cre, both of which are responsive to CCR (43). This plasticity allows CcpA to bind half-sites with altered sequences and provides an explanation for how CcpA can bind such a large number of degenerate cre elements that are found in different contexts throughout bacilli genomes (43, 44).


Figure 2
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  		FIGURE 2.
CcpA-(Crh-Ser46-P)-cre DNA contacts and flexibility of the DNA binding domain. A, schematic of the CcpA-cre contacts showing the average contacts to each half-site. The deoxyriboses of each nucleotide are numbered and shown as pentagons. Hydrogen bonds are indicated by arrows and van der Waals contacts by diamonds. Protein-DNA phosphate backbone interactions are depicted by arrows. NH indicates amide backbone contacts. B, stereoview of the C{alpha} superimposition of residues 60-330 of all DNA-bound CcpA structures. The N and C termini and the HTH regions are labeled. The arrow indicates the loop between {alpha}3 and the hinge helix that imparts flexible binding by the individual HTH elements.

 
CcpA-Crh-Ser46-P Interactions—Each Crh-Ser46-P monomer docks onto a CcpA subunit in a manner similar to that utilized by HPr-Ser46-P. Indeed, the primary interaction interface is forged between Crh-Ser46-P residues located on helices {alpha}1 and {alpha}2 and the turn between beta strand beta1 and helix {alpha}1 and the surface exposed region of CcpA that includes helices I and IX from the N-subdomain of CcpA. Thus, CcpA interaction interfaces of HPr-like proteins can be broken into two regions: contact regions I (CRI), encompassing residues 15-28 (helices {alpha}1 and the preceding loop) and contact region II (CRII), comprised primarily of {alpha}2 and phosphorylated residue Ser46 (Fig. 3).

Contact Region II: Conserved CcpA-HPr/Crh Interactions—Residues from CRII of Crh-Ser46-P include the phosphoresidue Ser46-P, which is located one residue N-terminal to {alpha}2, and the remainder of {alpha}2. Residues from this region are completely between HPr and Crh and in conserved to addition Ser46-P include Ile47, Met48and Met51, (Fig. 3, A and B). As in the CcpA-(HPr-Ser46-P)-cre structure, Crh-Ser46-P residue Ser46-P is contacted specifically by two CcpA basic residues, Arg303 and Lys307, which are located near the center of CcpA helix IX (Fig. 4A). This interaction is buttressed by Tyr89. CcpA cannot bind HPr-like proteins at physiologically relevant concentrations unless they are phosphorylated on residue Ser46 (6, 26, 34). Such phosphorylation of Ser46 increases the binding affinity of CcpA for cre DNA, indicating the critical nature of this interaction in both binding specificity and affinity (26, 34). In addition, several hydrophobic residues on {alpha}2 interact with hydrophobic residues on CcpA helix IX to form a water-free interface. These Crh-Ser4646-P residues, which are conserved in HPr, include Ile47, Met48, and Met51 and interact with CcpA residues Ala299, Val300, and Leu304 (Fig. 3B).

Contact Region I: Discrimination in the Binding of HPr-like Proteins to CcpA—CRI, consisting of the loop residues-{alpha} 1 motif, contains loop residues Gln15 Ala16 and {alpha}1 residues Arg17, Ala20 Val23and Gln24(Fig. 3)., Crh-Ser46-P residues Val23 and Gln24 make similar contacts with CcpA in the CcpA-(HPr-Ser46-P)-cre and CcpA-(Crh-Ser46-P)-cre structures. However, unlike CRII, in which all CcpA interacting residues are conserved between HPr and Crh, CRI contains two positions, 15 and 20, that differ between HPr and Crh. In all Crh proteins sequenced thus far these residues are glutamine and alanine, whereas in all HPr proteins they are histidine and threonine (Fig. 3A). In the CcpA-(HPr-Ser46-P)-cre structure, His15 of HPr hydrogen bonds to the chain of CcpA residue Asp296 (N{delta}1-O{delta}1, 2.5 Å), whereas the O{delta}2 of Asp296 engages in a hydrogen bond with the amide nitrogen of HPr residue Ala16. Just as the interactions between HPr CRII residue Ser46-P and CcpA basic residues Arg303 and Lys307 serve as key tethering points for HPr-Ser46-P CRII, the interactions between HPr-Ser46-P residues His15 and Ala16 and CcpA residue Asp296, serve as the anchor for HPr-Ser46-P contact region I to CcpA. In addition, these interactions function to partition the high energy CCR and low energy phosphoenolpyruvate:sugar phosphotransferase pathways because CcpA cannot bind HPr-His15-P and HPr-Ser46-P cannot bind the phosphoenolpyruvate:sugar phosphotransferase enzyme, E1 (45).


Figure 3
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  		FIGURE 3.
The CcpA-(Crh-Ser46-P) interface: CRI and II CRII. A, sequence alignment of known HPr and Crh proteins. The residues of B. subtilis HPr are numbered above the alignment. Protein and organisms are labeled on the left side of the alignment. Residues that are highly conserved in both HPr and Crh are boxed in black. Residues that are less well conserved between HPr and Crh are boxed in gray and residues that are conserved in Crh but differ from HPr are boxed in orange. Organisms and accession numbers are: HPr_Bsu (B. subtilis; P08877 [GenBank] ), HPr_Bme (B. megaterium; O69250 [GenBank] ), HPr_Bli (Bacillus licheniformis; Q65KB6), HPr_Bce (Bacillus cereus; Q731P9), HPr_Bha (Bacillus halodurans; Q9K8D2), Ban_HPr (Bacillus anthracis; Q81Ml0), HPr_Bth (Baccillus thuringiensis; Q9F166), HPr_Oih (Oceanobacillus iheyensis; Q8ENY1), Crh_Bsu (B. subtilis; O06976 [GenBank] ), Crh_Bme (B. megaterium; Footnote 3), Crh_Bli (B. licheniformis; Q65EH3), Crh_Bce (B. cereus; Q815J5), Crh_Bha (B. halodurans; Q9K8D2), Ban_Crh (B. anthracis; Q81X62, Crh_Bth (B. thuringiensis; Q6HBD7), Crh_Bha (B. halodurans; Q9K708), Crh_Oih (O. iheyensis; Q8ENL6). Shown below the alignment are the secondary structure elements of Crh with beta strands represented as arrows and {alpha} helices as rectangles. The secondary structural elements and loop region not involved in CcpA binding are colored black and gray, respectively. Strand beta5, which is present in HPr but absent in Crh, is shown as a gray arrow. Secondary structural elements located in contact regions I and II are colored green and red, respectively. Residues that interact with CcpA are identical between Crh-Ser46-P and HPr-Ser46-P are indicated by black asterisks under the alignment, whereas the two residues that are different between Crh and HPr and make different contacts to CcpA are indicated by orange asterisks. B, view of the CcpA-(Crh-Ser46-P) interface. One CcpA subunit is gray and Asp99' from the other CcpA subunit is blue. CcpA helices I and IX are labeled and Crh-Ser46-P contact regions I and II are colored green and red, as in Fig. 3A. Residues from the elements of each protein that interact are shown as colored sticks and labeled. C, superimposition of the loop-{alpha}1 contact regions of HPr-Ser46-P (yellow) and the three Crh-Ser46-P molecules in the asymmetric unit of the CcpA-(Crh-Ser46-P)-cre structure (blue, green, and red). The shortest distances (Å) between residue 15 of Crh and HPr and residues Asp296 and Arg324 of CcpA are indicated as solid lines of respective color.

 
We predicted that the conservative H15Q substitution should retain the key hydrogen bond with CcpA residue Asp296 (27). Surprisingly, only two of the three crystallographically independent CcpA-Crh-Ser46-P complexes show an interaction between Crh-Ser46-P residue Gln15 and CcpA residue Asp296. Moreover, these contacts are weak (between 3.5 and 4.5 Å). Indeed, the structure shows that the primary contact to Crh-Ser46-P residue Gln15 is from the side chain of CcpA residue Arg324(Figs. 3, B and C, and 4A). By contrast, in the CcpA-(HPr-Ser46-P)-cre complex, Arg324 is greater than 5.0 Å from His15(Fig. 3C). In the CcpA-(Crh-Ser46-P)-cre structure the Arg324 side chain rotates down into the CcpA-Crh interface to hydrogen bond with Gln15 (average contact distance of 3.1 Å) (Fig. 3C). Interestingly, sequence alignments show that Arg324 is conserved only in CcpA proteins from bacilli. This suggests that the Arg324-Gln15 interaction represents a bacillispecific CcpA-(Crh-Ser46-P) contact, which is supported further by the fact that Crh has, thus far, been identified only in bacilli. By contrast to the HPr-Ser46-P His15 interaction with CcpA residue Asp296, the interaction between Crh-Ser46-P residue Gln15 and CcpA residue Arg324 is far weaker. This is evidence by the long contact distances between Gln15 and Arg324 as well as the poorer electron density and higher B-factors for these residues in all independent CcpA-(Crh-Ser46-P)-cre structures. Interestingly, CcpA does not engage in a similar contact to His15 of HPr-Ser46-P, although Arg324 is free to do so. The underlying reason is unknown, but perhaps might originate in the residual positive electrostatic nature of His15, which would repel the guanidinium group of Arg324.


Figure 4
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  		FIGURE 4.
Structural basis for the differential binding of HPr-like proteins to CcpA. A, comparison of CcpA interactions with (HPr-Ser46-P) contact region I (left panel) and CcpA interactions with (Crh-Ser46-P) contact region I (right panel). Residues involved in protein-protein interactions are shown as sticks with dashed lines indicating favorable interactions. In the left panel, CcpA is colored blue and HPr cyan, whereas in the right panel, CcpA is colored red and Crh magenta. B, stereoview of the overlay of C{alpha} residues 60-330 of CcpA from the CcpA-(HPr-Ser46-P)-cre structure (blue) and the three CcpA subunits of the CcpA-(Crh-Ser46-P)-cre structure (red, magenta, and yellow) revealing the shift of contact region I of Crh-Ser46-P away from CcpA. Helices {alpha}1 and {alpha}2 from contact regions I and II are labeled.

 
The T20A substitution in Crh is the only other difference between the CcpA interacting residues of HPr-Ser46-P and Crh-Ser46-P (Fig. 3A). In the CcpA-(HPr-Ser46-P)-cre structure Thr20 hydrogen bonds to the phenolic side chain of CcpA residue Tyr295 46. Obviously, this contact is not possible in the CcpA-(Crh-Ser46-P)-cre complex. The last key CcpA interacting residue in CRI is Arg17. In the CcpA-(HPr-Ser46-P)-cre structure, the guanidinium side chain of Arg17 makes two contacts to residues Asp69' and Asp99', which are located on the other subunit of the CcpA dimer. These "cross-contacts" have been postulated to be important in the allosteric DNA-binding mechanism of CcpA. In the CcpA-(Crh-Ser46-P)-cre structure, the cross-contacts are weakened as Arg17 interacts with only side chain of Asp99' (Fig. 3B). Thus, the interactions between CcpA and CRII of both HPr-Ser46-P and Crh-Ser46-P are highly conserved, but the contacts between CcpA and the CRI of these CCR coregulators are different.

Basis for Differential Binding of Crh-Ser46-P and HPr-Ser46-P to CcpA—The CcpA-(Crh-Ser46-P)-cre structure demonstrates that although Crh-Ser46-P and HPr-Ser46-P bind CcpA in the same manner globally, there are several differences, specifically in the CRI-CcpA interface, that may explain the reduced affinity of Crh-Ser46-P for CcpA compared with HPr-Ser46-P (Fig. 4A). Underscoring these differences, superimpositions of the C{alpha} atoms of one CcpA subunit of the CcpA-(Crh-Ser46-P)-cre structure onto those of a CcpA subunit of the CcpA-(HPr-Ser46-P)-cre structure reveals that whereas the CRII of HPr and Crh dock nearly identically onto CcpA, the CRIs do not. Specifically, the CRI of Crh-Ser46-P is shifted away from the CcpA molecule as compared with the corresponding region of HPr-Ser46-P (Fig. 4B). This relocation results from the loosening of interactions between loop-{alpha}1 (CRI) residues of Crh-Ser46-P to CcpA, primarily the weaker Gln15-Arg324 contact, and the loss of the Thr20-Tyr295 hydrogen bond. The shift of CRI away from CcpA also explains the loss of the cross-contact from Arg17 to CcpA residue Asp69', which in this conformation is too far (>4 Å) to form a meaningful interaction. The different docking mode of Crh-Ser46-P is highlighted quantitatively by the buried surface area between CcpA and Crh-Ser46-P, which, whereas still sizeable, is 100 Å2 less than that observed between HPr-Ser46-P and CcpA (1300 Å2 compared with 1400 Å2). An additional contribution to the different binding affinities of Crh-Ser46-P and HPr-Ser46-P to CcpA may be the difference in the unliganded/liganded states of these phosphoproteins, if Crh actually exists in a monomer to dimer equilibrium in vivo. Indeed, we find that the domain-swapped Crh dimer would not be competent for CcpA binding. However, the physiological relevance, if any, of the domain-swapped Crh dimer remains to be determined.

Crh-Ser46-P Is a Functional Corepressor for CcpA—Binding of HPr-Ser46-P or Crh-Ser46-P to CcpA allows the regulator to bind cre sites with high affinity and specificity. The CcpA-(HPr-Ser46-P)-cre and apo-CcpA structures reveal that HPr-Ser46-P mediates a novel two-component allosteric DNA-binding activation mechanism that involves rotation of the CcpA subdomains as well as a direct, coregulator-induced relocation of a pivot-point residue, Thr61, located between the N-domain and the DNA-binding domain of CcpA (27). Key to the relocation of Thr61 is the interaction between Ser46-P and CcpA residue Arg303, which causes a rotation of Arg303 and a concomitant shift in the position of Tyr89. This movement is translated to the entire Tyr89 loop, which causes the hydrogen bond between Tyr91 and Thr306 to break. Tyr91 then rotates toward the CcpA dimer interface, dislodging Thr61 (27). The final result is the juxtaposition of the DNA-binding hinge regions, allowing formation of the hinge helices in the presence of cognate DNA. What is notable about this activation mechanism is the principal role played by residues of helix {alpha}2. Overlays of the CcpA-(Crh-Ser46-P)-cre and CcpA-(HPr-Ser46-P)-cre structures show that although {alpha}1 of the Crh complex is significantly shifted as compared with {alpha}1 in the HPr complex, {alpha}2 remains similarly docked against CcpA (Fig. 4B). Moreover, {alpha}2 residues of both Crh and HPr make essentially identical contacts to CcpA. Therefore, despite the weakening of the CRI interface, the interactions between the CRII of Crh-Ser46-P and CcpA remain adequately positioned to flip the DNA-binding switch to "on." This finding provides an atomic explanation for data showing that although Crh-Ser46-P binds CcpA with lower affinity, this phosphoprotein is fully capable of activating CcpA to bind DNA and hence to play a significant role in CCR.


Figure 5
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  		FIGURE 5.
Proximity of the putative small molecule adjunct corepressor binding pocket and the acidic C-terminal tail of Crh. The structure is depicted and colored as described in the legend to Fig. 1A except that the cre DNA is shown as a gray CPK model and one Crh-Ser46-P molecule is magenta. The last observed residue of each Crh-Ser46-P subunit is labeled C. The putative small molecule (Glu-6-P/Fru-6-P; G6P/FBP) binding pockets of each CcpA subunit are labeled.

 
Whereas HPr-Ser46-P and Crh-Ser46-P are clearly physiologically relevant corepressors for CcpA, recent data suggests that Glu-6-P and Fru-1,6-P may act as adjunct corepressors (34). These studies reveal that Glu-6-P and Fru-1,6-P can enhance the DNA binding of the CcpA-(HPr-Ser46-P) complex. Interestingly, however, they do not enhance DNA binding by the CcpA-(Crh-Ser46-P) complex. These results could be explained if Crh-Ser46-P binding somehow occludes the small molecule effector binding pocket of CcpA. Examination of the CcpA-(Crh-Ser46-P)-cre structure shows no obvious occlusion of the pocket by Crh-Ser46-P, however, the C terminus of Crh-Ser46-P is near the putative small molecule binding pocket of the other subunit of the CcpA dimer (Fig. 5). Intriguingly, the last few residues (81-84) of Crh-Ser46-P, which are acidic, are mostly disordered in the structure, suggesting the possibility that these residues could block the pocket or interfere electrostatically with binding of the negatively charged Fru-6-P and Glu-6-P. The C-terminal regions of Crh and HPr show the most structural divergence between the two proteins. Instead of the extended C-terminal coil found in Crh, HPr contains a short turn followed by beta5, which folds back into the core of HPr, effectively preventing blockage of the CcpA pocket by these residues. In addition, unlike Crh, the C terminus of HPr is not negatively charged. Although these contrasting properties of the C-terminal regions of HPr and Crh could explain the differential CcpA DNA-binding enhancement by Glu-6-P and Fru-6-P, additional data are clearly needed to understand the roles of these adjunct corepressors and the conditions under which they may function.

In conclusion, the structure of the CcpA-(Crh-Ser46-P)-cre complex reveals that Crh-Ser46-P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser46-P. The reduced affinity of Crh-Ser46-P for CcpA as compared with that of HPr-Ser46-P is explained readily by weaker Crh-Ser46-P interactions of its CRI to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix {alpha}2 (CRII) of Crh-Ser46-P is maintained and thereby demonstrates this contact region is necessary and sufficient to throw the allosteric switch and activate cre binding by CcpA. Finally, this study, which reveals how small differences in protein-protein interfaces can have a significant impact on the structure of a complex, underscores the need to take great caution in modeling protein-protein interactions even when a highly homologous structure is available.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1ZVV for CcpA-(Crh-Ser46-P)-cre ternary complex) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by a Burroughs Wellcome Career Development Award 992863 (to M. A. S.) and the Deutsche Forschungsgemeinschaft through SFB 473 and the Fonds der Chemischen Industrie (to W. H.). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the United States Department of Energy under contract DE-AC03-78SF00098 at the Lawrence Berkeley National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Robert A. Welch Distinguished University Chair in Chemistry. To whom correspondence should be addressed: Unit 1000, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-834-6390; Fax: 713-834-6397; E-mail: rgbrenna{at}mdanderson.org.

2 The abbreviations used are: CCR, carbon catabolite repression; cre, catabolite responsive element; CcpA, catabolite control protein A; Crh, catabolite responsive HPr; HPr, histidine containing protein; CRI, contact region I; CRII, contact region II; r.m.s., root mean square; HTH, helix turn helix. Back

3 M. Malton, personal communication. Back

4 M. A. Schumacher and R. G. Brennan, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank the Advanced Light Source and their support staff.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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