FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2090916175> ?p ?o ?g. }

• W2090916175 endingPage "32515" @default.
• W2090916175 startingPage "32508" @default.
• W2090916175 abstract "Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5Å reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35Å between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model. Enzyme I (EI) is the phosphoenolpyruvate (PEP)-protein phosphotransferase at the entry point of the PEP-dependent sugar phosphotransferase system, which catalyzes carbohydrate uptake into bacterial cells. In the first step of this pathway EI phosphorylates the heat-stable phospho carrier protein at His-15 using PEP as a phosphoryl donor in a reaction that requires EI dimerization and autophosphorylation at His-190. The structure of the full-length protein from Staphylococcus carnosus at 2.5Å reveals an extensive interaction surface between two molecules in adjacent asymmetric units. Structural comparison with related domains indicates that this surface represents the biochemically relevant contact area of dimeric EI. Each monomer has an extended configuration with the phosphohistidine and heat-stable phospho carrier protein-binding domains clearly separated from the C-terminal dimerization and PEP-binding region. The large distance of more than 35Å between the active site His-190 and the PEP binding site suggests that large conformational changes must occur during the process of autophosphorylation, as has been proposed for the structurally related enzyme pyruvate phosphate dikinase. Our structure for the first time offers a framework to analyze a large amount of research in the context of the full-length model. Group translocation is the membrane transport mechanism by which a substrate is chemically modified to an impermeable derivative as it crosses the cell membrane. This energy-efficient transport strategy is used by bacteria for the uptake of rapidly metabolizable sugars, and it is achieved through a highly conserved three component phospho-relay system called the phosphoenolpyruvate:sugar phosphotransferase system (PTS) 4The abbreviations used are: PTS, PEP-dependent sugar phosphotransferase system; PEP, phosphoenolpyruvate; EI and EII, Enzymes I and II; HPr, heatstable histidine phospho carrier protein; EIN, N-terminal protease-resistant domain; EIC, protease-sensitive C-terminal domain; P-His, phosphohistidine; PPDK, pyruvate phosphate dikinase; CPK, Corey-Pauling-Koltun; MAD, multiple-wavelength anomalous dispersion; DTPA-BMA, diethylene-triaminepentaacetic acid bismethylamide. 4The abbreviations used are: PTS, PEP-dependent sugar phosphotransferase system; PEP, phosphoenolpyruvate; EI and EII, Enzymes I and II; HPr, heatstable histidine phospho carrier protein; EIN, N-terminal protease-resistant domain; EIC, protease-sensitive C-terminal domain; P-His, phosphohistidine; PPDK, pyruvate phosphate dikinase; CPK, Corey-Pauling-Koltun; MAD, multiple-wavelength anomalous dispersion; DTPA-BMA, diethylene-triaminepentaacetic acid bismethylamide. (1Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 2Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (301) Google Scholar, 3Kundig W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (322) Google Scholar). The PTS catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to a sugar while it is being transported across the membrane. It consists of two universal components, Enzyme I (molecular mass, 63 kDa) (4Ginsburg A. Peterkofsky A. Arch. Biochem. Biophys. 2002; 397: 273-278Crossref PubMed Scopus (32) Google Scholar, 5Chauvin F. Brand L. Roseman S. Res. Microbiol. 1996; 147: 471-479Crossref PubMed Scopus (41) Google Scholar), hereafter referred to as EI, and the heat-stable histidine phospho carrier protein (HPr) (molecular mass, 9 kDa), and in addition several membrane-associated components, which are sugar-specific and are collectively designated as Enzyme II (EII) complexes (3Kundig W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (322) Google Scholar). The PTS cascade starts with the autophosphorylation of EI on a conserved histidine (His-190 in the Staphylococcus carnosus EI studied in this report) in a reaction that uses PEP as phosphoryl donor (4Ginsburg A. Peterkofsky A. Arch. Biochem. Biophys. 2002; 397: 273-278Crossref PubMed Scopus (32) Google Scholar). Subsequently, the phosphoryl group is transferred to His-15 of the HPr protein, and ultimately to the imported hexose, in a series of transphosphorylation reactions mediated by the components of the sugar-specific EII complex. The PTS is not only responsible for sugar uptake; it also represents a major sensing and signaling system in the bacterial cell. The phosphorylation state of the components of the PTS pathway is directly coupled to the regulation of carbohydrate metabolism, chemotaxis toward carbon sources (6Saier Jr., M.H. Reizer J. Mol. Microbiol. 1994; 13: 755-764Crossref PubMed Scopus (171) Google Scholar), carbon catabolite repression (7Warner J.B. Lolkema J.S. Microbiol. Mol. Biol. Rev. 2003; 67: 475-490Crossref PubMed Scopus (196) Google Scholar, 8Saier 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), and nitrogen metabolism (9Reizer J. Reizer A. Saier Jr., M.H. Jacobson G.R. Protein Sci. 1992; 1: 722-726Crossref PubMed Scopus (63) Google Scholar). Because EI catalyzes the first step in the pathway and because its activity levels will determine the phosphorylation state of the downstream PTS components, it may play a key regulatory role in the control of PTS and its downstream metabolic effects (10Weigel N. Kukuruzinska M.A. Nakazawa A. Waygood E.B. Roseman S. J. Biol. Chem. 1982; 257: 14477-14491Abstract Full Text PDF PubMed Google Scholar, 11Patel H.V. Vyas K.A. Mattoo R.L. Southworth M. Perler F.B. Comb D. Roseman S. J. Biol. Chem. 2006; 281: 17579-17587Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 12Patel H.V. Vyas K.A. Savtchenko R. Roseman S. J. Biol. Chem. 2006; 281: 17570-17578Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). EI is highly conserved throughout bacteria, displaying a high degree of sequence similarity among different species (13Reizer J. Hoischen C. Reizer A. Pham T.N. Saier Jr., M.H. Protein Sci. 1993; 2: 506-521Crossref PubMed Scopus (44) Google Scholar). It consists of an N-terminal protease-resistant portion (EIN, residues 1-264) and a protease-sensitive C-terminal domain (EIC, residues 265-573) (4Ginsburg A. Peterkofsky A. Arch. Biochem. Biophys. 2002; 397: 273-278Crossref PubMed Scopus (32) Google Scholar). The N-terminal region is responsible for HPr binding, whereas the C-terminal region binds PEP (14Oberholzer A.E. Bumann M. Schneider P. Bachler C. Siebold C. Baumann U. Erni B. J. Mol. Biol. 2005; 346: 521-532Crossref PubMed Scopus (33) Google Scholar). The EIN domain consists of two subdomains: an α-helical domain and an α/β domain, called the phospho-histidine (P-His) domain, that contains the intermediate phosphoacceptor His-190 (in S. carnosus) (15Liao D.I. Silverton E. Seok Y.J. Lee B.R. Peterkofsky A. Davies D.R. Structure. 1996; 4: 861-872Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Garrett D.S. Seok Y.J. Liao D.I. Peterkofsky A. Gronenborn A.M. Clore G.M. Biochemistry. 1997; 36: 2517-2530Crossref PubMed Scopus (152) Google Scholar). The EIN and the HPr protein form a stable complex that has been studied by NMR, showing that interactions between HPr and EI occur through the helical domain (from now on termed the HPr-binding domain), which is responsible for substrate specificity. This interaction places the phosphoacceptor His-15 of HPr and the His-190 of EI at optimal separation for efficient phosphotransfer (17Garrett D.S. Seok Y.J. Peterkofsky A. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1999; 6: 166-173Crossref PubMed Scopus (207) Google Scholar). The C-terminal domain of EI displays sequence similarity with the PEP-binding domain of the pyruvate phosphate dikinase (PPDK) enzyme (18Herzberg O. Chen C.C. Kapadia G. McGuire M. Carroll L.J. Noh S.J. Dunaway-Mariano D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2652-2657Crossref PubMed Scopus (130) Google Scholar, 19Herzberg O. Chen C.C. Liu S. Tempczyk A. Howard A. Wei M. Ye D. Dunaway-Mariano D. Biochemistry. 2002; 41: 780-787Crossref PubMed Scopus (34) Google Scholar). This enzyme catalyzes the reversible conversion of ATP, pyruvate, and inorganic phosphate (Pi) into PEP, AMP, and pyrophosphate through a phosphoryl-enzyme intermediate, and it has a phosphohistidine acceptor domain structurally equivalent to that of EI. Erni and coworkers (14Oberholzer A.E. Bumann M. Schneider P. Bachler C. Siebold C. Baumann U. Erni B. J. Mol. Biol. 2005; 346: 521-532Crossref PubMed Scopus (33) Google Scholar) have recently obtained the crystal structure of the C-terminal domain of Thermoanaerobacter tengcongensis EI showing that the overall fold is very similar, and the configuration of the active site is almost identical to that of the PEP-binding domain from PPDK. Both PPDK and the PEP-binding region of T. tengcongensis crystallized as dimers, and in both cases the dimerization interface involves equivalent regions of the PEP-binding domains. On the other hand, biochemical studies have shown that EI exists in a monomer-dimer equilibrium (20Chauvin F. Toptygin D. Roseman S. Brand L. Biophys. Chem. 1992; 44: 163-173Crossref PubMed Scopus (6) Google Scholar, 21Dimitrova M.N. Szczepanowski R.H. Ruvinov S.B. Peterkofsky A. Ginsburg A. Biochemistry. 2002; 41: 906-913Crossref PubMed Scopus (18) Google Scholar) where only the dimeric form is competent for autophosphorylation (22Chauvin F. Brand L. Roseman S. J. Biol. Chem. 1994; 269: 20263-20269Abstract Full Text PDF PubMed Google Scholar). Those studies showed that dimer formation is stimulated by PEP and magnesium ions but that the interconversion between monomer and dimer is very slow, suggesting that oligomerization may be the rate-limiting step for the activation of EI and as a consequence may determine the activity of the PTS pathway and its downstream effects (21Dimitrova M.N. Szczepanowski R.H. Ruvinov S.B. Peterkofsky A. Ginsburg A. Biochemistry. 2002; 41: 906-913Crossref PubMed Scopus (18) Google Scholar, 22Chauvin F. Brand L. Roseman S. J. Biol. Chem. 1994; 269: 20263-20269Abstract Full Text PDF PubMed Google Scholar). More recently Roseman and coworkers (11Patel H.V. Vyas K.A. Mattoo R.L. Southworth M. Perler F.B. Comb D. Roseman S. J. Biol. Chem. 2006; 281: 17579-17587Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 12Patel H.V. Vyas K.A. Savtchenko R. Roseman S. J. Biol. Chem. 2006; 281: 17570-17578Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) using ultracentrifugation experiments demonstrated that the presence of PEP and magnesium induce conformational changes in both the monomeric and dimeric forms of EI. Nevertheless, it remains unclear how dimerization activates the autocatalytic potential of EI and more generally how the different catalytic centers distributed in different domains of the protein interact during the reaction cycle. We present here the first crystal structure of the full-length EI from S. carnosus at a resolution of 2.5 Å, which for the first time reveals the spatial arrangement of the three protein domains. Aspects of dimer formation and stabilization as well as implications for catalysis will be discussed. Protein Expression and Purification—EI from S. carnosus was overexpressed in Escherichia coli (strain DH5α) harboring the plasmid pUC-ptsO2.6X (23Kohlbrecher D. Eisermann R. Hengstenberg W. J. Bacteriol. 1992; 174: 2208-2214Crossref PubMed Google Scholar). Cells were grown in the presence of isopropyl-β-d-thiogalactoside in TBY broth (10 g of Tryptone, 5 g of NaCl, 5 g of yeast extract/liter). Cells (13 g of wet weight from 6 liters of culture) were suspended in 25 ml of standard buffer (50 mm Tris-HCl, pH 7.5, 0.1 mm dithiothreitol, 0.1 mm EDTA, 0.1 mm phenylmethanesulfonyl fluoride), disrupted by sonication, and centrifuged at 25,000 × g. The supernatant was applied to a Q-Sepharose column (120 ml) using a gradient of 600 ml (0-0.6 m NaCl in standard buffer). EI was detected by SDS-PAGE (10% acrylamide) at 0.5 m NaCl. The enzyme pool was adjusted to 20% ammonium sulfate, applied to a butyl-TSK column (420 ml, Tosohaas, Montgomeryville, PA) and eluted with a linear gradient of 2 liters of 20-0% ammonium sulfate in standard buffer. EI eluted at 5% ammonium sulfate and was pure according to native (pH 9) and denaturing (SDS) PAGE. Standard yields were 40-50 mg of protein. Prior to crystallization experiments the protein was passed through a Sephadex G-25 column equilibrated with 50 mm HEPES, pH 6.5, and concentrated in Centricon tubes (Millipore) to a concentration of 20 mg/ml. The storage solution was supplemented with PEP and MgCl2, both to a final concentration of 5 mm. Crystallization—Crystals of full-length EI were obtained by the vapor diffusion method. Initially hanging drops made with 1 μl of protein, 1 μl of crystallization buffer (30% polyethylene glycol 4000, 0.2 m Li2SO4, and 0.1 m Tris-HCl, pH 8.5), and 0.2 μl of additive solution (solution 11 of the Hampton Crystal Screen, Hampton Research: 0.1 m trisodium citrate dehydrate, pH 5.6, 1 m ammonium dihydrogen phosphate) were equilibrated against 500 μl of crystallization buffer in standard Linbro crystallization plates. Hexagonal crystals appeared after 1-2 days and diffracted typically to 7.0 Å in a synchrotron beam. After 5-7 days new monoclinic crystals appeared in the drops while the hexagonal crystals tended to disappear. These crystals diffracted typically to 2.5 Å with a synchrotron x-ray source. Initially the presence of solution 11 of the Hampton Crystal Screen in the crystallization mixture was the result of an accidental contamination, however this proved to be essential for the reproducibility of the crystals. A second crystallization condition was obtained using sitting drops and 30% (w/v) polyethylene glycol 4000, 0.2 sodium malonate, and 0.1 m Tris-HCl, pH 8.5, as crystallization buffer. This second condition did not require additives and produced only monoclinic crystals with similar symmetry properties and diffraction power. For data collection the crystals were soaked in crystallization buffer containing 7.5% (v/v) polyethylene glycol 400 as cryoprotectant and flash-cooled by immersion in liquid nitrogen. Data Collection, Structure Determination, and Refinement—All diffraction data were collected under a cryogenic stream at 100 K. Preliminary characterization was performed at beamlines ID14-1 and ID29 of the European Synchrotron Radiation Facility and beamline BW7a of the Deutsches Elektronen-Synchroton. EI crystals obtained in the presence of sodium malonate were soaked for 3 h in crystallization buffer supplemented with 0.1 m of the Gadolinium complex Gd·DTPA-BMA (24Girard E. Stelter M. Anelli P.L. Vicat J. Kahn R. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 118-126Crossref PubMed Scopus (25) Google Scholar) and used in a multiple-wavelength anomalous dispersion (MAD) experiment conducted at beamline BM14 of the European Synchrotron Radiation Facility (Table 1). Data processing and scaling were done with the program suite XDS (25Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3200) Google Scholar).TABLE 1Summary of the crystallographic analysisNativePeak (λ1)Inflection (λ2)Remote (λ3)Space groupC2C2Unit cell dimensions (Å, °)a = 173.36, b = 46.85, c = 85.29, α = γ = 90, β = 101.464a = 177.34, b = 47.39, c = 86.61, α = γ = 90, β = 101.804Data collectionx-ray sourceESRF, ID14EH1ESRF, BM14Wavelength (Å)0.93401.72201.71090.976Resolution (Å)40-2.5045.7-2.7645.7-2.7645.73-2.5Highest shell (Å)2.6-2.52.90-2.762.90-2.762.90-2.76No. unique reflectionsaNumbers in parentheses refer to the highest resolution shell.23,054 (2,444)33,642 (3,785)33,251 (3,454)44,998 (2,834)Multiplicity7.152.41.791.9I/σ(I)aNumbers in parentheses refer to the highest resolution shell.21.9 (6.8)15.4 (3.04)14.5 (2.92)18.0 (6.7)RmeasbRedundancy-independent R factor as implemented in XDS (25). (%)aNumbers in parentheses refer to the highest resolution shell.6.1 (28.3)5.8 (33.3)5.1 (31.4)4.1 (13.4)Completeness (%)aNumbers in parentheses refer to the highest resolution shell.97.4 (93.9)88.8 (77.3)93.6 (70.6)97.7 (94.4)PhasingResolution (Å)45.7-2.8Figure-of-merit0.59RefinementResolution (Å)40-2.5No. of reflections (completeness)22,479 (95.0%)RworkcAs defined in CNS, the free R factor (Rfree) was calculated with 5% of reflections randomly omitted from the structure refinement (27). (%)0.22RfreecAs defined in CNS, the free R factor (Rfree) was calculated with 5% of reflections randomly omitted from the structure refinement (27). (%)0.27Protein atoms4,071SO4 ions1Water molecules172Root mean square deviationsBond length0.006Angles1.2a Numbers in parentheses refer to the highest resolution shell.b Redundancy-independent R factor as implemented in XDS (25Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3200) Google Scholar).c As defined in CNS, the free R factor (Rfree) was calculated with 5% of reflections randomly omitted from the structure refinement (27Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-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 (16909) Google Scholar). Open table in a new tab The coordinates of the heavy atoms and the initial set of phases were obtained with the program SOLVE (26Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar). These phases and the atomic model of the α-helical subdomain of the N-terminal region of EI (amino acids 20-142, PDB code 1ZYM) were used for phased molecular replacement using the program CNS (27Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-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 (16909) Google Scholar), which produced a solution for the α-helical N-terminal domain that agreed with the density map. Phase combination and density modification with the CNS program resulted in an improved electron density map, which allowed model extension. The structure of the PPDK (1DIK) (18Herzberg O. Chen C.C. Kapadia G. McGuire M. Carroll L.J. Noh S.J. Dunaway-Mariano D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2652-2657Crossref PubMed Scopus (130) Google Scholar) was superimposed on a partial model of the C-terminal portion and subsequently adjusted in the experimental electron density map. The structural model was completed based on the revised sequence of the S. carnosus enzyme, 5R. Rosenstein and F. Götz, (University of Tübingen, Tübingen, Germany) personal communication. in alternate rounds of model building and refinement using CNS (27Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-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 (16909) Google Scholar) and O (28Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (503) Google Scholar). In the later stages of model building, structure completion was continued with data collected from another crystal grown under the original conditions for the monoclinic form. This crystal was soaked in (NH4)2Os4Br6, which improved diffraction data but did not lead to a heavy atom derivative and was thus considered as a native crystal (Table 1). A large electron density peak in the PEP-binding region of the C-terminal domain, showing interactions with the side chains of two arginine and one asparagine residue compatible with a tetrahedral coordination sphere, indicated the presence of a phosphate or sulfate ion. Given that LiSO4 (0.2 m) is a component of the crystallization mixture, this peak was modeled as a sulfate ion. A summary of the crystallographic analysis is given in Table 1. Structural visualizations were done with the programs MOLSCRIPT along with Raster3D (Figs. 1B, 2, and 3) (29Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar, 30Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3865) Google Scholar) and PYMOL (Figs. 1A, 1C, and 4A). 6W. L. DeLano (2002) DeLano Scientific, San Carlos, CA, www.pymol.org. The atomic coordinates have been deposited with the Protein Data Bank (www.rcsb.org), under accession code 2HRO.FIGURE 2The C-terminal domain (EIC) and the PEP binding site. Stereo view of the PEP binding site from S. carnosus EI and the PPDK superimposed. Residues involved in binding and catalysis are represented as sticks. Side-chain carbon atoms and the corresponding residue numbers are displayed in green for EI and in gray for PPDK. The sulfate ion present in the EI, and the substrate analogue phosphonopyruvate from the PPDK structure are displayed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3The EI dimer. A, ribbon diagram of the EI dimer with a color scheme as in Fig. 1. The sulfate ions at the PEP binding sites are depicted as CPK models. The spatial separation of the P-His and PEP-binding domains is clearly visible. B, ribbon diagram of the dimeric C-terminal region of EI from S. carnosus (left) and T. tengcongensis (right) showing the structural conservation of the dimer interaction region. Double arrow lines have been drawn along the interaction surfaces to help identify the two monomers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Model of the conformational changes during catalysis. A, modeling the transition to the autophosphorylating conformation. Full-length EI as found in the crystal structure is represented with the different domains colored as in Fig. 1A. The relative position of the P-His-190-binding (cyan) and HPr-binding (red) domains after superposition with the structure of Z. mays PPDK (44Nakanishi T. Nakatsu T. Matsuoka M. Sakata K. Kato H. Biochemistry. 2005; 44: 1136-1144Crossref PubMed Scopus (23) Google Scholar) are presented. The arrow indicates the direction of movement that would be required to evolve from the former to the latter conformation. Steric hindrance between the HPr-binding domain and the C-terminal region indicates that the HPr-binding and P-His domains might slightly detach during this part of the reaction. His-190 is represented as CPK model. The sulfate ion at the PEP binding site is shown as CPK. B, a model for the conformational changes associated with the catalytic cycle of EI. The C-terminal domain is represented in green, the P-His domain in blue, the HPr-binding domain in red, and the linker regions in gray. The HPr protein is represented in light green. Yellow dots mark the location of the reaction centers: the PEP binding site, the His-190 of the P-His domain and the acceptor His-15 of HPr. The phosphoryl group that is transferred from PEP to HPr is represented by a red dot. Panel I represents the structure as observed in the crystal (only one monomer is shown). Once PEP is bound to EIC, P-His must approach EIC (IIa). This possible intermediate conformation is suggested by the structure of Z. mays PPDK (44Nakanishi T. Nakatsu T. Matsuoka M. Sakata K. Kato H. Biochemistry. 2005; 44: 1136-1144Crossref PubMed Scopus (23) Google Scholar) and would require only small conformational changes at the level of the linker helix that could act like a lever arm. Autophosphorylation at His-190 would require binding of PEP to the C-terminal region and a closer contact between the P-His and the C-terminal domains. This rearrangement would probably require a slight displacement of the HPr-binding domain (IIb). After completion of the autophosphorylation cycle the C-terminal and P-His domains dissociate, and the HPr-binding domain (with HPr bound) adopts a tight conformation with respect to the P-His domain (III). In the scheme, HPr is shown as entering the cycle at the last stage (III), but association during other stages cannot be excluded.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structural Organization of EI—EI from S. carnosus (23Kohlbrecher D. Eisermann R. Hengstenberg W. J. Bacteriol. 1992; 174: 2208-2214Crossref PubMed Google Scholar) was overexpressed and purified as described under “Experimental Procedures.” Initial hexagonal crystals appearing in Hampton Standard screens diffracted poorly, whereas monoclinic crystals, appearing 3-5 days later, diffracted up to 2.5 Å. For phase determination a novel gadolinium compound (24Girard E. Stelter M. Anelli P.L. Vicat J. Kahn R. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 118-126Crossref PubMed Scopus (25) Google Scholar) was particularly useful, because it allowed straightforward recording of a full data set using MAD that was used for structure determination as described under “Experimental Procedures.” The resulting electron density map showed one molecule per asymmetric unit and was easy to interpret in the regions corresponding to the HPr-binding and the PEP-binding domains. However, the region containing the P-His domain (residues 4-22 and 156-229) showed weak electron density even at the later stages of refinement. Despite this, 70 of the 102 Cα atoms in this region were assigned, which is enough to identify the fold and determine its orientation relative to the other two domains; however, many of the side chains of this domain, including His-190, were not visible and were modeled as alanines. A stereo view of an electron density map calculated with the final model but omitting the P-His domain (to exclude any possibility of phase bias in this region) is presented in Fig. 1A. The electron density corresponding to the backbone of the P-His domain is clearly visible. The final model (Rwork/Rfree: 22/27%, see Table 1) consists of 551 residues, 1 sulfate ion, and 172 water molecules. 22 of 573 residues in the full-length protein were not visible in the electron density: 3 and 2 residues at the N- and C-termini, respectively, and 17 in the P-His domain. The full-length EI monomer is composed of three structurally independent domains separated by long linkers (Fig. 1B). It shows an extended conformation, ∼110 Å in length with a maximum width of ∼60 Å. The N-terminal region (EIN) and the PEP-binding C-terminal domain (EIC) are separated by a long (40 Å) and easily accessible linker helix (residues 233-260, in gray in Fig. 1, B and C), which explains the proteolytic sensitivity of EI (32LiCalsi C. Crocenzi T.S. Freire E. Roseman S. J. Biol. Chem. 1991; 266: 19519-19527Abstract Full Text PDF PubMed Google Scholar, 33Lee B.R. Lecchi P. Pannell L. Jaffe H. Peterkofsky A. Arch. Biochem. Biophys. 1994; 312: 121-124Crossref PubMed Scopus (28) Google Scholar). The HPr-binding and the P-His domain (depicted in red and blue, respectively, in Fig. 1) are also separated by two extended linker regions (depicted in gray) with only few interactions between them. The P-His domain (residues 4-22 and 156-229) contains the His-190, the initial acceptor of the phosphoryl group from PEP (15Liao D.I. Silverton E. Seok Y.J. Lee B.R. Peterkofsky A. Davies D.R. Structure. 1996; 4: 861-872Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This region of the structure showed weak electron density and high temperature factors (see above), suggesting that it is flexible in our crystals, presumably due to a lack of interactions with the other domains that could stabilize its position. This conformational flexibility may have functional implications, as discussed below. The HPr-binding domain (red in Fig. 1) is inserted in the β1-β2 loop region of the P-His domain. It spans amino acids" @default.
• W2090916175 created "2016-06-24" @default.
• W2090916175 creator A5014022014 @default.
• W2090916175 creator A5031277712 @default.
• W2090916175 creator A5043028388 @default.
• W2090916175 creator A5044282246 @default.
• W2090916175 creator A5068760266 @default.
• W2090916175 creator A5083195785 @default.
• W2090916175 date "2006-10-01" @default.
• W2090916175 modified "2023-10-12" @default.
• W2090916175 title "Structure of the Full-length Enzyme I of the Phosphoenolpyruvate-dependent Sugar Phosphotransferase System" @default.
• W2090916175 cites W1480792688 @default.
• W2090916175 cites W1517629137 @default.
• W2090916175 cites W1521025514 @default.
• W2090916175 cites W1567485884 @default.
• W2090916175 cites W1582831987 @default.
• W2090916175 cites W1647410008 @default.
• W2090916175 cites W1958844676 @default.
• W2090916175 cites W1965277349 @default.
• W2090916175 cites W1972035895 @default.
• W2090916175 cites W1982552688 @default.
• W2090916175 cites W1989979669 @default.
• W2090916175 cites W1995017064 @default.
• W2090916175 cites W1995340751 @default.
• W2090916175 cites W2000840258 @default.
• W2090916175 cites W2001367916 @default.
• W2090916175 cites W2010610895 @default.
• W2090916175 cites W2012574818 @default.
• W2090916175 cites W2017124784 @default.
• W2090916175 cites W2021461291 @default.
• W2090916175 cites W2023180023 @default.
• W2090916175 cites W2028231353 @default.
• W2090916175 cites W2030111609 @default.
• W2090916175 cites W2041968999 @default.
• W2090916175 cites W2043551811 @default.
• W2090916175 cites W2044387929 @default.
• W2090916175 cites W2052527181 @default.
• W2090916175 cites W2059721778 @default.
• W2090916175 cites W2061860586 @default.
• W2090916175 cites W2071320074 @default.
• W2090916175 cites W2073060468 @default.
• W2090916175 cites W2075183488 @default.
• W2090916175 cites W2075451080 @default.
• W2090916175 cites W2076183099 @default.
• W2090916175 cites W2076326298 @default.
• W2090916175 cites W2078574004 @default.
• W2090916175 cites W2080528351 @default.
• W2090916175 cites W2093086129 @default.
• W2090916175 cites W2099630547 @default.
• W2090916175 cites W2130394726 @default.
• W2090916175 cites W2140520515 @default.
• W2090916175 cites W2141883257 @default.
• W2090916175 cites W2154297643 @default.
• W2090916175 cites W86972446 @default.
• W2090916175 cites W1533386780 @default.
• W2090916175 doi "https://doi.org/10.1074/jbc.m513721200" @default.
• W2090916175 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16867985" @default.
• W2090916175 hasPublicationYear "2006" @default.
• W2090916175 type Work @default.
• W2090916175 sameAs 2090916175 @default.
• W2090916175 citedByCount "27" @default.
• W2090916175 countsByYear W20909161752012 @default.
• W2090916175 countsByYear W20909161752013 @default.
• W2090916175 countsByYear W20909161752014 @default.
• W2090916175 countsByYear W20909161752015 @default.
• W2090916175 countsByYear W20909161752016 @default.
• W2090916175 countsByYear W20909161752017 @default.
• W2090916175 countsByYear W20909161752019 @default.
• W2090916175 countsByYear W20909161752021 @default.
• W2090916175 countsByYear W20909161752022 @default.
• W2090916175 crossrefType "journal-article" @default.
• W2090916175 hasAuthorship W2090916175A5014022014 @default.
• W2090916175 hasAuthorship W2090916175A5031277712 @default.
• W2090916175 hasAuthorship W2090916175A5043028388 @default.
• W2090916175 hasAuthorship W2090916175A5044282246 @default.
• W2090916175 hasAuthorship W2090916175A5068760266 @default.
• W2090916175 hasAuthorship W2090916175A5083195785 @default.
• W2090916175 hasBestOaLocation W20909161751 @default.
• W2090916175 hasConcept C143937172 @default.
• W2090916175 hasConcept C181199279 @default.
• W2090916175 hasConcept C185592680 @default.
• W2090916175 hasConcept C2777108408 @default.
• W2090916175 hasConcept C2777504003 @default.
• W2090916175 hasConcept C45656491 @default.
• W2090916175 hasConcept C55493867 @default.
• W2090916175 hasConceptScore W2090916175C143937172 @default.
• W2090916175 hasConceptScore W2090916175C181199279 @default.
• W2090916175 hasConceptScore W2090916175C185592680 @default.
• W2090916175 hasConceptScore W2090916175C2777108408 @default.
• W2090916175 hasConceptScore W2090916175C2777504003 @default.
• W2090916175 hasConceptScore W2090916175C45656491 @default.
• W2090916175 hasConceptScore W2090916175C55493867 @default.
• W2090916175 hasIssue "43" @default.
• W2090916175 hasLocation W20909161751 @default.
• W2090916175 hasOpenAccess W2090916175 @default.
• W2090916175 hasPrimaryLocation W20909161751 @default.
• W2090916175 hasRelatedWork W1518355276 @default.
• W2090916175 hasRelatedWork W1682509349 @default.

• next