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The Structural Basis of Signal Transduction for the Response Regulator PrrA from Mycobacterium tuberculosis

2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês

10.1074/jbc.m512004200

1083-351X

Elżbieta Nowak, Santosh Panjikar, Petr V. Konarev, Dmitri I. Svergun, Paul A. Tucker,

Bacteriophages and microbial interactions

The structure of the two-domain response regulator PrrA from Mycobacterium tuberculosis shows a compact structure in the crystal with a well defined interdomain interface. The interface, which does not include the interdomain linker, makes the recognition helix and the trans-activation loop of the effector domain inaccessible for interaction with DNA. Part of the interface involves hydrogen-bonding interactions of a tyrosine residue in the receiver domain that is believed to be involved in signal transduction, which, if disrupted, would destabilize the interdomain interface, allowing a more extended conformation of the molecule, which would in turn allow access to the recognition helix. In solution, there is evidence for an equilibrium between compact and extended forms of the protein that is far toward the compact form when the protein is inactivated but moves toward a more extended form when activated by the cognate sensor kinase PrrB. The structure of the two-domain response regulator PrrA from Mycobacterium tuberculosis shows a compact structure in the crystal with a well defined interdomain interface. The interface, which does not include the interdomain linker, makes the recognition helix and the trans-activation loop of the effector domain inaccessible for interaction with DNA. Part of the interface involves hydrogen-bonding interactions of a tyrosine residue in the receiver domain that is believed to be involved in signal transduction, which, if disrupted, would destabilize the interdomain interface, allowing a more extended conformation of the molecule, which would in turn allow access to the recognition helix. In solution, there is evidence for an equilibrium between compact and extended forms of the protein that is far toward the compact form when the protein is inactivated but moves toward a more extended form when activated by the cognate sensor kinase PrrB. The use of regulatory systems to sense and respond to changing environmental conditions is an intrinsic feature that enables bacteria to survive and adapt to a variety of external challenges. Two-component signaling (TCS) 2The abbreviations used are: TCS, two-component signaling; PDB, Protein Data Bank; SAXS, small x-ray angle scattering; r.m.s., root mean square; r.m.s.d., r.m.s. deviation. 2The abbreviations used are: TCS, two-component signaling; PDB, Protein Data Bank; SAXS, small x-ray angle scattering; r.m.s., root mean square; r.m.s.d., r.m.s. deviation. systems are the principle mechanism used by bacteria to perform this task (1Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington, D. C.1995Crossref Google Scholar). A typical TCS consists of a sensor histidine kinase and a response regulator (RR). Histidine kinases are usually membrane-anchored proteins with a characteristic core consisting of a histidine-containing dimerization domain and a catalytic domain. In response to extracellular, and in a few cases, intracellular, conditions, the histidine kinase autophosphorylates at a histidine residue, usually in the dimerization domain, by phosphotransfer from the catalytic (ATPase) domain of the adjacent protomer. It then acts as a phosphodonor to a universally conserved aspartic acid residue in the response regulator. RRs typically consist of two domains with the N-terminal (receiver) domain being the phosphoacceptor domain and the C-terminal domain being the effector. The effector domain is, in most cases, DNA binding and is involved in transcriptional regulation of the genes necessary to respond to the sensed environment. Phosphorylation of the aspartic acid residue located in the receiver domain activates the effector domain in a manner that is still incompletely understood, not least because there is little structural information on full-length response regulators and no structural information on activated full-length (i.e. multidomain) response regulators. TCSs have been identified as potential antibacterial targets because they play a key role in controlling cellular processes (2Barret J.F. Hoch J.A. Antimicrob. Agents Chemother. 1998; 42: 1529-1536Crossref PubMed Google Scholar). The lack of these systems in higher eukaryotes makes them potentially selective and unique antibacterial drug targets, There is, however, little biochemical information available on the TCSs of pathogenic bacteria such as Mycobacterium tuberculosis (MtB). The H37Rv strain of MtB has 12 putative TCSs including the recently discovered Rv3220-Rv1626 pair (3Morth J.P. Gosmann S. Nowak E. Tucker P.A. FEBS Lett. 2005; 579: 4145-4148Crossref PubMed Scopus (36) Google Scholar) as well as five putative orphan response regulator and sensor kinase proteins. The PrrA-PrrB pair (Genome locii Rv0903c-Rv0902c) is one of five TCS gene pairs conserved in all mycobacterial species (4Tyaga J.S. Sharma D. Curr. Sci. (Bangalore). 2004; 86: 93-102Google Scholar) and can consequently be assumed to play (a) fundamental role(s) in mycobacteria. The function and signals sensed by TCS proteins in M. tuberculosis are still poorly characterized despite their apparent importance, although some initial findings have been made (5Betts J.C. Lukey P.T. Robb L.C. McAdam R.A. Duncan K. Mol. Microbiol. 2002; 43: 717-731Crossref PubMed Scopus (1100) Google Scholar, 6Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (630) Google Scholar, 7Rodriguez G.M. Voskuil M.I. Gold B. Schoolnik G.K. Smith I. Infect. Immun. 2002; 70: 3371-3381Crossref PubMed Scopus (380) Google Scholar, 8Zhart T.C. Deretic V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12706-12709Crossref PubMed Scopus (180) Google Scholar). The PrrA-PrrB TCS has been implicated in the early intracellular multiplication of M. tuberculosis (9Ewann F. Jackson M. Pethe K. Cooper A. Mielcarek N. Ensergueix D. Gicquel B. Locht C. Supply P. Infect. Immun. 2002; 70: 2256-2263Crossref PubMed Scopus (81) Google Scholar, 10Ewann F. Locht C. Supply P. Microbiology (Reading). 2004; 150: 241-246Crossref PubMed Scopus (35) Google Scholar). The cDNAs corresponding to co-transcripts from the PrrA-PrrB locus have been recovered from MtB cultured in human blood monocyte-derived macrophages but not from cells cultured in standard laboratory medium, indicating that the genes are transcribed in response to host-cell interaction (11Graham E.J. Clark-Curtiss E.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11554-11559Crossref PubMed Scopus (386) Google Scholar). Thus the ability to survive within a host cell makes these TCS proteins prime targets for the development of new antibacterial agents. Response regulators all contain structurally similar N-terminal domains with a β1-α1-β2-α2-β3-α3-β4-α4-β5-α5 topology that contains the conserved phosphorylation site. They can be subdivided into families depending upon the expected structure of the C-terminal, or effector, domain. The three families, which can also have subdivisions, are named after representative proteins, namely OmpR/PhoB, NarL/FixJ, and NtrC/DctD. The first two classes are σ70-dependent activators, whereas the last family consist of σ54-dependent activators and contains an additional central ATPase domain necessary for open complex formation of the RNA polymerase. The RR studied here (PrrA) belongs to the OmpR/PhoB family. OmpR and PhoB are members of the wingedhelix-turn-helix family of DNA-binding proteins (12Brennan R.G. Cell. 1993; 74: 773-776Abstract Full Text PDF PubMed Scopus (216) Google Scholar). There are, however, important structural differences between OmpR and PhoB, for example, in the length of interdomain linker and in the transactivation loop that proceeds the DNA recognition helix (13Okamura H. Hanaoka S. Nagadoi A. Makino K. Nishimura Y. J. Mol. Biol. 2000; 295: 1225-1236Crossref PubMed Scopus (80) Google Scholar). Structural studies investigating individual receiver domains of response regulators in their active and inactive forms provide some information about conformational changes occurring upon phosphorylation, but how this change is transmitted to activate the C-terminal effector domain is, in most cases, still a mystery. This is because structural information is much more limited in the case of intact proteins and totally absent on full-length proteins in the activated form. Consequently, there is a paucity of knowledge about interactions between the regulatory and effector domains. At the time of writing, there are only five structures available on full-length RRs in the Protein Data Bank (PDB). Two of them, DrrB (14Robinson V.L. Wu T. Stock A.M. J. Bacteriol. 2003; 185: 4186-4194Crossref PubMed Scopus (88) Google Scholar) and DrrD (15Buckler D.R. Zhou Y. Stock A.M. Structure (Camb.). 2002; 10: 153-164Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), belong to the OmpR/PhoB subfamily and were crystallized from Thermatoga maritima. Although it is often the case that proteins from thermophilic bacteria are more amenable to crystallization than their mesophilic counterparts (15Buckler D.R. Zhou Y. Stock A.M. Structure (Camb.). 2002; 10: 153-164Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), we present here the structure of PrrA from M. tuberculosis. We show that although in the crystal it exhibits a compact conformation, inhibitory for DNA binding, in solution, there is probably a percentage of a more open conformation that would be more favorable for DNA binding. The PrrA gene (genomic location tag Rv0903c) was cloned by PCR from genomic M. tuberculosis (H37Rv strain) DNA. DNA flanking primers 5′-TATACCATGGGCGGCATGGACACTGGTGTG-3′ and 5′-GATTATCACTGCA TACGCAGCACGAATCCG-3′were engineered to provide melting temperatures of ∼65 °C with extensions encoding NcoI and XhoI restriction sites and ligated with T4 DNA ligase into a pETM11 (EMBL) expression vector with an N-terminal His6 tag and a recognition sequence for tobacco etch virus protease. Following sequence confirmation, the plasmid was transformed into Rosetta(DE3)pLysS cells. Cells were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside at A600 = 0.7 and grown for 5 h at 25 °C. The cells were harvested by centrifugation and lysed by sonication in a buffer containing 50 mm Tris·HCl, 200 mm NaCl, 5 mm β-mercaptoethanol, pH 7.5 (buffer A), and two protease inhibitor tablets (Roche Applied Science). The lysate was spun down and loaded onto a 5-ml nickel (HiTrap) column equilibrated in buffer A. The protein was eluted with a 100-ml linear gradient of 10-300 mm imidazole in buffer A. The fractions containing PrrA were pooled and diluted to a final imidazole concentration of 50 mm. The protein was then mixed with tobacco etch virus protease in a 1:20 ratio and left at 4 °C overnight to cleave the His6 tag. The digested protein (which has an additional Gly and Ala at the N terminus resulting from the cloning procedure) was applied to the HiTrap column equilibrated in buffer A containing 50 mm imidazole. The flow-through fractions were collected, concentrated to a final volume of 5 ml, and further purified on HiLoad 26/60 Superdex 75 equilibrated in 20 mm Tris·HCl, 100 mm NaCl, 2 mm dithiothreitol, pH 7.5 (buffer B). The peak fractions were concentrated using a 10-kDa cut-off centricon (Vivaspin) to a concentration of 12 mg/ml as measured by Bradford's assay (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Crystallization and X-ray Data Collection—Initial crystallization conditions were identified using the sparse matrix Cryo screen from Hampton Research. Following optimization, the best crystals were obtained after 1 week by hanging drop vapor diffusion, in which 2 μl of protein were mixed with 2 μl of reservoir buffer, containing 130 mm calcium acetate, 15% polyethylene glycol 8000, 20% glycerol, and 100 mm sodium cacodylate at pH 6.5. To obtain crystals containing Mg2+ in the active site, we substituted calcium acetate by magnesium acetate in the crystallization buffer. The crystals grew as intertwined clusters, making it necessary to break them to obtain single crystal fragments. For data collection, crystals were directly transferred from the drop and flashed-cooled to 100 K in the cold gas stream. Measurements were carried out on the X11 and X13 EMBL beam lines at Deutsches Elektronen Synchrotron (DESY) Hamburg using a MAR165 mm CCD detector. Diffraction images were processed using DENZO, and the intensities were scaled and reduced using SCALEPACK (17Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) and converted to structure amplitudes using the program TRUNCATE within the CCP4 program suite (18Collaborative Computational Project Number Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). Crystals (calcium-containing) belong to space group P1 with unit cell parameters a = 37.2, b = 46.5, c = 65.7 Å and α = 100.3, β = 101.7, γ = 92.5 ° with two molecules in the asymmetric unit and an estimated solvent content of 48%. Data collection details are summarized in Table 1.TABLE 1Data collection and refinement statisticsPrrA—calciumPrrA—magnesiumData collectionaNumbers given in brackets are from the last resolution shell.BeamX13X11Wavelength (Å)0.810.81Space groupP1P1Cell parameters (Å)a = 37.2, b = 46.5,a = 37.4, b = 46.5,c = 65.7c = 65.6(°)α = 100.3, β = 101.7,α = 100.3, β = 101.0,γ = 92.5γ = 92.9Resolution (Å)1.771.58No. of unique reflections39,82353,033Completeness (%)98.3 (72.0)96.5 (85.5)Multiplicity2.2 (2.2)4.1 (3.7) /σ 17.6 (2.0)22.7 (2.5)Rsym (%)bRsym=∑hkl∑i|I1(hkl)-〈I(hkl)〉|/∑hkl∑iIi(hkl), where Ii is the intensity of the ith reflection and is the average intensity.4.8 (57.4)6.5 (71.2)RefinementResolution (Å)20-1.7720-1.58RworkcRwork=∑hkl{|Fo|-|Fc|}/∑hkl|Fo|, where Fo is the observed structure factor and Fc is the calculated factor. / RfreedRfree was calculated as for Rwork but on 5% of the reflections excluded from the refinement. (%)18.8/24.818.6/22.8Number of residues467468Missing residues1918Number of waters340203Number of Tris molecules1Number of glycerol molecules12Number of acetate ions2r.m.s.d. bonds (Å)0.0180.017r.m.s.d. angles (°)1.801.88Ramachandran plotMost favored (%)89.690.4Additional allowed (%)8.68.4Generously allowed (%)1.30.8Disallowed (%)0.50.5a Numbers given in brackets are from the last resolution shell.b Rsym=∑hkl∑i|I1(hkl)-〈I(hkl)〉|/∑hkl∑iIi(hkl), where Ii is the intensity of the ith reflection and is the average intensity.c Rwork=∑hkl{|Fo|-|Fc|}/∑hkl|Fo|, where Fo is the observed structure factor and Fc is the calculated factor.d Rfree was calculated as for Rwork but on 5% of the reflections excluded from the refinement. Open table in a new tab Structure Determination—The sequence with accession code Q10531 was used for homology modeling with the SWISS-PROT server, which is based on an automated comparative protein modeling (19Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4376) Google Scholar). The resulting model was derived from the PDB entries 1KGS (15Buckler D.R. Zhou Y. Stock A.M. Structure (Camb.). 2002; 10: 153-164Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), 1MVO (20Birck C. Chen Y. Hulett F.M. Samama J-P. J. Bacteriol. 2003; 185: 254-261Crossref PubMed Scopus (46) Google Scholar), 1NXW, 1NXV, and 1NXP (21Bent C.J. Isaacs N.W. Mitchell T.J. Riboldi-Tunnicliffe A. J. Bacteriol. 2004; 186: 2872-2879Crossref PubMed Scopus (44) Google Scholar). The N- and C-terminal domains of the homology model were placed in two coordinate files containing residues 7-119 and 138-223, respectively. Residues 120-137 were omitted on the assumption that the interdomain linker would be flexible. The model of the N-terminal domain was used initially as the search model for molecular replacement, with data in the range 10-4 Å used in the rotation and translation function searches. Two molecules per asymmetric unit were expected on the basis of the Matthews parameter (22Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7894) Google Scholar), and consequently, a multiple copy search for the domain was performed using the program MOLREP (23Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4098) Google Scholar). The R-factor and correlation coefficient for the first domain were 0.56 and 0.20, respectively, which became 0.55 and 0.22, respectively, upon location of the domain in the second molecule. The resultant model containing both N-terminal domains was then fixed, and the C-terminal domain was used as a search model. Again, a multiple copy search was performed for two domains; however, only one was easily found dropping the R-factor to 0.49 and increasing the correlation coefficient 0.43. The C-terminal domain of the second molecule was created based on the relative orientations of the N- and C-terminal domains of the first molecule and the complete solution was checked in molecular modeling program O (24Jones T.A. Zhou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar) for good packing contacts before proceeding to refinement. The model was then subjected for rigid body refinement in CNS (25Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. 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), considering each domain as a separate entity. Further positional and B-factor refinement were performed using CNS when the R-factor and Rfree (26Brünger A.T. Methods Enzymol. 1997; 277: 366-396Crossref PubMed Scopus (270) Google Scholar) dropped to 0.43 and 0.47, respectively. At this stage, sigmaa-weighted maps were calculated using the program SIGMAA (27Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2035) Google Scholar), and careful examination of the maps allowed corrections to be incorporated into the model. Finally, the model was used as an initial model for automated model building using the program ARP/wARP (28Perrakis A. Morris R.M. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar) against the 1.77 Å data. 96% of the model was correctly built in 10 building cycles. Further refinement was continued using REFMAC5 (29Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). The model was iteratively improved by combination of refinement and manual building using Coot (30Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22534) Google Scholar). In the final stages of refinement, a bulk solvent correction, non-crystallographic symmetry restraints, and anisotropic scaling were used in REFMAC. The refinement was monitored throughout using the R-free, which was calculated using 5% of the unique reflections. In the final model of PrrA, complexed with either Ca2+ or Mg2+, two N-terminal residues (Gly and Ala) introduced during the cloning procedure together with the first six residues of the protein are not visible in the electron density and, because they extend into the solvent region, are assumed to be disordered. Other regions with uninterpretable electron density are residues 87-90 of molecule A (PrrA-Ca and PrrA-Mg), Arg-88 of the molecule B (PrrA-Ca and PrrA-Mg), residues Gly-214 and Gly-215 of molecule A (PrrA-Ca), and Gly-215 and Ser-128 of molecule B (PrrA-Mg). The overall geometric quality of the model was assessed using PROCHECK (31Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). 89.6% and 90.4% of the amino acid residues, for PrrA-Ca and Prra-Mg, respectively, were found in the most favorable region of the Ramachandran plot with one residue (Val-63) in the disallowed region. The refined coordinates and the structure factors for the PrrA structure complexed with Ca2+ and Mg2+ have been deposited with the RCSB (PDB ID: 1YS6 and 1YS7, respectively). The figures have been produced with Molscript (32Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), CCP4MG, and WebLab Viewer 2.0. SAXS Experiments and Data Analysis—The synchrotron radiation x-ray scattering data were collected on the X33 camera (33Koch M.H.J. Bordas J. Nucl. Instr. and Meth. 1983; 208: 461-469Crossref Scopus (287) Google Scholar, 34Boulin C.J. Kempf R Gabriel A. Koch M.H.J. Nucl. Instr. and Meth. A. 1998; 269: 312-320Crossref Scopus (232) Google Scholar) of the EMBL (on the DORIS III storage ring at DESY). Solutions of PrrA were measured at several protein concentrations (from 2 to 13 mg/ml using a MAR345 two-dimensional image plate detector (Marr Research), in the range 0.12 < s < 4.5 nm-1. To check for radiation damage, two successive 2-min exposures were compared, but no radiation effects were observed. The data were averaged after normalization to the intensity of the incident beam, corrected for the detector response, and the scattering of the buffer was subtracted. The difference data were extrapolated to zero solute concentration following standard procedures. The PrrBdependent activation was studied in solutions containing 4 mg/ml PrrA and 0.1 mg/ml PrrBHCD(see Ref. 35Nowak E. Panjikar S. Morth J.P. Jordanova R. Svergun D.I. Tucker P.A. Structure (Camb.). 2005; 14: 275-285Abstract Full Text Full Text PDF Scopus (38) Google Scholar for the nomenclature) with 5 mm Mg2+ and 10 mm ATP. The scattering from the PrrBHCD/Mg2+/ATP solution was then subtracted as background. All data manipulations were performed using the program package PRIMUS (36Konarev P.V. Volkov V.V. Sokolova A.V. Koch M.H.J. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2275) Google Scholar). The forward scattering I(0) and the radius of gyration Rg were evaluated by the Guinier approximation (37Guinier A. Ann. Phys. (Paris). 1939; 12: 161-231Google Scholar) and by using the indirect transform package GNOM (38Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2875) Google Scholar). The molecular masses of the solutes were evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (molecular mass = 66 kDa) and were compatible with monomeric PrrA in solution. The scattering from the high resolution models was calculated using the program CRYSOL (39Svergun D.I. Barberato C. Koch M.H.J. J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2688) Google Scholar) Given the atomic coordinates, the program fits the experimental intensity I(s) by adjusting the excluded volume of the particle and the contrast of the hydration layer surrounding the particle in solution to minimize the discrepancy χ2=1N-1∑jIexp(sj)-clcalc(sj)σ(sj)2(Eq. 1) where N is the number of experimental points, Iexp(s), Icalc(s), and σ(sj) are the experimental and calculated intensity and the experimental error at the momentum transfer sj, respectively, and c is a scaling factor. Rigid body modeling of PrrA was done by freely rotating its C-terminal domain around the residue Ser-128 and computing the fits to the experimental data with CRYSOL. The volume fractions of the component in the mixtures best fitting the small x-ray angle scattering (SAXS) data from inactivated and activated PrrA were evaluated using the computed scattering curves from crystallographic models of PrrA, DrrB, and DrrD by the program OLIGOMER (36Konarev P.V. Volkov V.V. Sokolova A.V. Koch M.H.J. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2275) Google Scholar). PrrA crystallizes in the triclinic space group P1 with two molecules in the asymmetric unit. The structure of the protein was solved by molecular replacement and refined at 1.77 Å resolution when complexed with Ca2+(referred to as PrrA-Ca) and at 1.58 Å resolution when complexed with Mg2+ (referred to as PrrA-Mg). The structure of molecule B from PrrA-Mg is shown in Fig. 1, which shows the N-terminal receiver domain and C-terminal effector domain connected by a linker region. The receiver domain has the expected (α/β)5 topology, with five parallel strands β2-β1-β3-β4-β5 forming the hydrophobic core surrounded by two helices (α1 and α5) on one side and three (α2-α4) on the other. The C-terminal domain has a wingedhelix fold and is composed of a four-stranded antiparallel β-sheet followed by a three-helix bundle and a C-terminal β-hairpin. The two domains are connected by a long, well ordered linker between α5 and β6 and pack together, forming an extensive buried interface. The two molecules in the asymmetric unit superimpose with an r.m.s.d. of 0.56 Å (for 204 Cα atoms). The largest difference is observed in the linker region (Fig. 2) and the region containing the α4 helix, which have r.m.s.d. values of 8.0 and 8.8 Å, respectively.FIGURE 2A stereo schematic showing the superimposition of the two independent molecules in the asymmetric unit of PrrA-Ca. Molecule A is colored red, and molecule B is colored blue. Differences between the calcium- and magnesium-bound structures are small, and details are described under "Results and Discussion."View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Receiver Domain—The active site of PrrA is defined by a crevice formed by loops β1-α1 and β3-α3 and contains all residues essential for phosphorylation. These are Asp-14, Asp-15, Asp-58, and Lys-108 (Fig. 3). In some response regulators, Asp-14 is replaced by glutamate. Bacterial response regulators are phosphorylated by the cognate sensor kinase in a divalent cation-dependent reaction, and divalent ions are usually observed in the catalytic site. Ca2+ and Mg2+ metal (depending on crystallization conditions) ions were found in the Asp-15/Asp-58 pocket (Fig. 3B). Both ions exhibit octahedral coordination, interacting in the same way with the carboxylate groups of Asp-15 and Asp-58 as well as the carbonyl oxygen of Asn-60. The three remaining coordination sites are occupied by water molecules. The distances between Ca2+ and Mg2+ and their coordinating atoms lie in the ranges 2.20-2.54 and 1.96-2.14 Å, respectively, in good agreement with equivalent distances in other protein structures (40Gouet P. Fabry B. Guillet. V. Brick C. Mourey L. Kahn D. Samama J.P. Structure (Camb.). 1999; 7: 1517-1526Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The amino group of conserved Lys-108 forms a salt bridge to the carboxylate groups of Asp-58 (2.9-3.1 Å) and Asp-14 (2.6-2.8 Å). Asp-14 is not directly involved in coordinating the metal ion but forms a strong hydrogen bond to a water molecule of the octahedral metal coordination sphere. Similar coordination of the metal ion has been observed for CheY (41Bellsolell L. Prieto J. Serrano L. Coll M. J. Mol. Biol. 1994; 238: 489-495Crossref PubMed Scopus (118) Google Scholar). The electron density for the region of the protein surrounding the metal coordination site is mostly well defined, in contrast to the case in DivK (42Guillet V. Ohta N. Cabantous S. Newton A. Samama J-P. J. Biol. Chem. 2002; 277: 42003-42010Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), where a destabilizing effect on the protein structure was observed due to metal binding, which resulted in the disordering of the α4-β4 region. The absence of electron density for residues 87-90 in molecule A of the PrrA-Ca structure is unlikely to result from metal binding because the same region in molecule B with metal bound in the active site is well ordered. Ser-86 is one of the residues known to be crucial in signal transduction (43Appleby J.L. Bourret R.B. J. Bacteriol. 1998; 180: 3563-3569Crossref PubMed Google Scholar), although in the other response regulators of the OmpR/PhoB family, it is normally threonine (Fig. 4). This residue has a slightly different environment in each of the crystallographically independent molecules of PrrA, and this environment also differs slightly when comparing the Mg2+ - and Ca2+-containing structures. In molecule A, the carbonyl group of Ser-86 forms a water-mediated interaction with the main chain nitrogen of Lys-108, whereas in molecule B, the carbonyl oxygen of Ser-86 and main chain nitrogen of Lys-108 are directly hydrogen-bonded (2.7 Å). In addition, for molecule B, the hydroxyl oxygen of Ser-86 makes a salt-bridge interaction with the side chain of Arg-94 in both PrrA-Mg and PrrA-Ca. In molecule A of PrrA-Mg, the serine hydroxyl group is hydrogen-bonded to the carbonyl oxygen of Ala-96, whereas the region around Ser-86 in molecule A of PrrA-Ca is not well ordered and is not discussed further. The side chain of the highly conserved Tyr-105, which is another key residue in signal transduction (44Zhu X. Rebello J. Matsumura P. Volz K. J. Biol. Chem. 1997; 272: 5000-5006Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), forms a very strong hydrogen bond with the side-chain oxygen of Asn-198 (2.2-2.5 Å) in molecule A of PrrA-Ca and PrrA-Mg, as well as in molecule B of PrrA-Ca. The hydroxyl group of Tyr-105 for molecule B of PrrA-Mg hydrogen-bonds the main-chain nitrogen of Asn-198 and the amino group of a Tris molecule. One noticeable difference between the two molecules in the asymmetric unit is the length of helix α4, which is unwound in molecule