Phosphoprotein Crh-Ser46-P Displays Altered Binding to CcpA to Effect Carbon Catabolite Regulation
2005; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m509977200
ISSN1083-351X
AutoresMaria A. Schumacher, Gerald Seidel, Wolfgang Hillen, Richard G. Brennan,
Tópico(s)Protein Structure and Dynamics
ResumoIn 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 α 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. 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 α 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. Carbon catabolite repression/regulation (CCR) 2The 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.2The 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. is a global regulatory mechanism utilized by bacteria to select, out of a mixture of compounds, the carbon source providing the optimal growth advantage (1Saier Jr., M.H. Chauvaux S. Deutscher J. Reizer J. Ye J.J. Trends Biochem. Sci. 1995; 20: 267-271Abstract Full Text PDF PubMed Scopus (143) Google Scholar, 2Stülke J. Hillen W. Annu. Rev. Microbiol. 2000; 54: 849-880Crossref PubMed Scopus (286) Google Scholar, 3Bruckner R. Titgemeyer F. FEMS Microbiol. Lett. 2002; 209: 141-148Crossref PubMed Scopus (348) Google Scholar). 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) (4Wray L.V. Pettengill F.K. Fisher S.H. J. Bacteriol. 1994; 176: 1894-1902Crossref PubMed Google Scholar, 5Henkin T.M. FEMS Microbiol. Lett. 1996; 135: 9-15Crossref PubMed Google Scholar, 6Jones B.E. Dossonnet V. Küster E. Hillen W. Deutscher J. Klevit R.E. J. Biol. Chem. 1997; 272: 26530-26535Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 7Miwa Y. Nagura K. Eguchi S. Fukuda H. Deutscher J. Fujita Y. Mol. Microbiol. 1997; 23: 1203-1213Crossref PubMed Scopus (69) Google Scholar, 8Turinsky A.J. Grundy F.J. Kim J.H. Chambliss G.H. Henkin T.M. J. 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J. Biol. Chem. 1992; 267: 15869-15874Abstract Full Text PDF PubMed Google Scholar). 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 (15Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (331) Google Scholar, 16Schumacher M.A. Choi K.Y. Lu F. Zalkin H. Brennan R.G. Cell. 1995; 83: 147-155Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 17Bell C.E. Lewis M. Nat. Struct. Biol. 2000; 7: 209-214Crossref PubMed Scopus (6) Google Scholar, 18Bell C.E. Lewis M. J. Mol. Biol. 2001; 312: 921-926Crossref PubMed Scopus (69) Google Scholar, 19Lewis M. Chang G. Horton N.C. Kercher M.A. Pace H.C. Schumacher M.A. Brennan R.G. Lu P. Science. 1996; 271: 1247-1254Crossref PubMed Scopus (647) Google Scholar, 20Choi K.Y. Zalkin H. J. Bacteriol. 1992; 174: 6207-6214Crossref PubMed Google Scholar). Although some studies suggest that glucose 6-phosphate and fructose 1,6-bisphosphate act as corepressors of CcpA, these data are not unequivocal (21Gösseringer R. Küster E. Galinier A. Deutscher J. Hillen W. J. Mol. Biol. 1997; 266: 665-676Crossref PubMed Scopus (122) Google Scholar, 22Kim J-H. Voskuil M.I. Chambliss G.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9590-9595Crossref PubMed Scopus (63) Google Scholar, 23Miwa Y. Fujita Y. J. Bacteriol. 2001; 183: 5877-5884Crossref PubMed Scopus (43) Google Scholar). By contrast, multiple studies have demonstrated that the Ser46-phosphorylated form of HPr binds CcpA as a corepressor, activating CcpA to bind cre sites (6Jones B.E. Dossonnet V. Küster E. Hillen W. Deutscher J. Klevit R.E. J. Biol. Chem. 1997; 272: 26530-26535Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 24Fujita Y. Miwa Y. Galinier A. Deutscher J. Mol. Microbiol. 1995; 17: 953-960Crossref PubMed Scopus (190) Google Scholar, 25Galinier A. Deutscher J. Martin-Verstraete I. J. Mol. Biol. 1999; 286: 307-314Crossref PubMed Scopus (100) Google Scholar, 26Aung-Hilbrich L.M. Seidel G. Wagner A. Hillen W. J. Mol. Biol. 2002; 319: 77-85Crossref PubMed Scopus (32) Google Scholar). The recent structure of the CcpA-(HPr-Ser46-P)-cre revealed the mechanism by which HPr-Ser46-P functions as a corepressor for CcpA (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 (15Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (331) Google Scholar, 16Schumacher M.A. Choi K.Y. Lu F. Zalkin H. Brennan R.G. Cell. 1995; 83: 147-155Abstract Full Text PDF PubMed Scopus (95) Google Scholar). In addition to HPr, a structural and functional homologue, Crh (for catabolite repression HPr) has been identified, but only in bacilli (28Galinier A. Haiech J. Kilhoffer M.C. Jaquinod M. Stülke J. Deutscher J. Martin-Verstraete I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8439-8444Crossref PubMed Scopus (208) Google Scholar). 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 (29Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 30Stülke J. Hillen W. J. Mol. Biol. 1998; 303: 545-553Google Scholar). 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 (28Galinier A. Haiech J. Kilhoffer M.C. Jaquinod M. Stülke J. Deutscher J. Martin-Verstraete I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8439-8444Crossref PubMed Scopus (208) Google Scholar, 31Poncet S. Mijakovic I. Nessler S. Gueguen-Chaignon V. Chaptal V. Galinier A. Boel G. Maze A. Deutscher J. Biochim. Biophys. Acta. 2004; 1697: 123-135Crossref PubMed Scopus (47) Google Scholar). 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 (28Galinier A. Haiech J. Kilhoffer M.C. Jaquinod M. Stülke J. Deutscher J. Martin-Verstraete I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8439-8444Crossref PubMed Scopus (208) Google Scholar, 32Darbon E. Galinier A. Le Coq D. Deutscher J. J. Mol. Microbiol. Biotechnol. 2001; 3: 439-444PubMed Google Scholar). 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 (33Gorke B. Fraysse L. Galinier A. J. Bacteriol. 2004; 186: 2992-2995Crossref PubMed Scopus (24) Google Scholar). 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 (33Gorke B. Fraysse L. Galinier A. J. Bacteriol. 2004; 186: 2992-2995Crossref PubMed Scopus (24) Google Scholar). 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 (34Seidel G. Diel M. Fuchsbauer N. Hillen W. FEBS Lett. 2005; 272: 2566-2577Crossref PubMed Scopus (44) Google Scholar), thereby indicating differences in their binding mechanisms. 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 (35Juy M. Penin F. Favier A. Galinier A. Montserret R. Haser R. Deutscher J. Bockmann A. J. Mol. Biol. 2003; 332: 767-776Crossref PubMed Scopus (35) Google Scholar, 36Favier A. Brutscher B. Blackledge M. Galinier A. Deutscher J. Penin F. Marion D. J. Mol. Biol. 2002; 317: 131-144Crossref PubMed Scopus (40) Google Scholar). 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 (34Seidel G. Diel M. Fuchsbauer N. Hillen W. FEBS Lett. 2005; 272: 2566-2577Crossref PubMed Scopus (44) Google Scholar). 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. 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 34Seidel G. Diel M. Fuchsbauer N. Hillen W. FEBS Lett. 2005; 272: 2566-2577Crossref PubMed Scopus (44) Google Scholar). 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The B. megaterium and B. subtilis Crh proteins (both 88 residues) share 64% sequence identity but their predicted CcpA interacting surfaces share 100% sequence identity 3M. Malton, personal communication.. 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 β = 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).TABLE 1Selected crystallographic statistics of the CcpA-(Crh-Ser46-P)-cre complexData setResolution (Å)68.70 - 2.96Overall Rsym(%)aRsym = ∑∑|I(h,k,l)-I(h,k,l)|/∑I(h,k,l), where I(h,k,l,)(j) is observed intensity and I(h,k,l) is the final average value of intensity.10.8 (38.7)bValues in parentheses are for the highest resolution shell.Overall I/σ(I)5.6 (1.7)Total reflections (No.)73,770Unique reflections (No.)31,223Completeness (%)95.8 (95.7)RefinementRwork/Rfree(%)cRwork = ∑∥Fobs|-|Fcalc∥/∑|Fobs| and Rfree ∑∥Fobs|-|Fcalc∥/∑|Fobs|; where all reflections belong to a test set of 5% randomly selected data.23.2/29.8r.m.s. deviationBond angles (°)1.51Bond lengths (Å)0.009B-values (Å2)2.70Ramachandran analysisMost favored (%/No.)81.1/895Additionally allowed (%/No.)17.2/195Generously allowed (%/No.)1.3/14Disallowed (%/No.)0.4/4a Rsym = ∑∑|I(h,k,l)-I(h,k,l)|/∑I(h,k,l), where I(h,k,l,)(j) is observed intensity and I(h,k,l) is the final average value of intensity.b Values in parentheses are for the highest resolution shell.c Rwork = ∑∥Fobs|-|Fcalc∥/∑|Fobs| and Rfree ∑∥Fobs|-|Fcalc∥/∑|Fobs|; where all reflections belong to a test set of 5% randomly selected data. Open table in a new tab 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), as a search model and the program EPMR (37Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (688) Google Scholar). 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 (38Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar, 39Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Crosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). This was followed by multiple cycles of simulated annealing and positional/thermal parameter refinement in CNS and rebuilding in O (38Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar, 39Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Crosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 (40Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) 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) (15Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. Science. 1994; 266: 763-770Crossref PubMed Scopus (331) Google Scholar, 16Schumacher M.A. Choi K.Y. Lu F. Zalkin H. Brennan R.G. Cell. 1995; 83: 147-155Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 17Bell C.E. Lewis M. Nat. Struct. Biol. 2000; 7: 209-214Crossref PubMed Scopus (6) Google Scholar, 18Bell C.E. Lewis M. J. Mol. Biol. 2001; 312: 921-926Crossref PubMed Scopus (69) Google Scholar, 19Lewis M. Chang G. Horton N.C. Kercher M.A. Pace H.C. Schumacher M.A. Brennan R.G. Lu P. Science. 1996; 271: 1247-1254Crossref PubMed Scopus (647) Google Scholar). The DNA-binding domain can be divided into two regions, α1 through α3, which form the HTH containing three-helix bundle, and α4, 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 Cα 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 Cα atoms. The larger deviation in superimposition of the dimer reflects the flexible attachment between helix α 3 and the hinge helix, α4 (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) (6Jones B.E. Dossonnet V. Küster E. Hillen W. Deutscher J. Klevit R.E. J. Biol. Chem. 1997; 272: 26530-26535Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). 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 Cα atoms. Crh-Ser46-P contains three α-helices (α1, residues 18-27; α2, residues 47-52; α3, residues 70-81) and four β-strands (β1, residues 2-6; β2, residues 32-37; β3, residues 41-44; β4, 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 Cα 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 α3, which is tilted slightly differently in the two proteins and the second is the absence of HPr-Ser46-P β strand β5 (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 (36Favier A. Brutscher B. Blackledge M. Galinier A. Deutscher J. Penin F. Marion D. J. Mol. Biol. 2002; 317: 131-144Crossref PubMed Scopus (40) Google Scholar). 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 β strand, β1, exchanges subunits (35Juy M. Penin F. Favier A. Galinier A. Montserret R. Haser R. Deutscher J. Bockmann A. J. Mol. Biol. 2003; 332: 767-776Crossref PubMed Scopus (35) Google Scholar). Superimposition of the Cα atoms of our Crh-Ser46-P structure onto the corresponding Cα 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. 4M. A. Schumacher and R. G. Brennan, manuscript in preparation.What 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 (34Seidel G. Diel M. Fuchsbauer N. Hillen W. FEBS Lett. 2005; 272: 2566-2577Crossref PubMed Scopus (44) Google Scholar). 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 (41Kalodimos C.G. Bonvin A.M. Salinas R.K. Wechselberger R. Boelens R. Kaptein R. EMBO J. 2002; 21: 2866-2876Crossref PubMed Scopus (98) Google Scholar, 42Kalodimos C.G. Biris N. Bonvin A.M. Levandoski M.M. Guennuegues M. Boelens R. Kaptein R. Science. 2004; 305: 350-352Crossref PubMed Scopus (423) Google Scholar). 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 (27Schumacher M.A. Allen G.S. Diel M. Seidel G. Hillen W. Brennan R.G. Cell. 2004; 118: 731-741Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) (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° (15Schumacher M.A. Choi K.Y. Zalkin H. Brennan R.G. 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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 α3, rotate as a unit about a flexible loop that links helices α3 and α4. 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
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