Crystal structure of a cyanobacterial sucrose‐phosphatase in complex with glucose‐containing disaccharides
2007; Wiley; Volume: 68; Issue: 3 Linguagem: Inglês
10.1002/prot.21481
ISSN1097-0134
AutoresSonia Fieulaine, John E. Lunn, Jean‐Luc Ferrer,
Tópico(s)Hemoglobin structure and function
ResumoProteins: Structure, Function, and BioinformaticsVolume 68, Issue 3 p. 796-801 Structure NoteFree Access Crystal structure of a cyanobacterial sucrose-phosphatase in complex with glucose-containing disaccharides Sonia Fieulaine, Sonia Fieulaine Institut de Biologie Structurale J-P. Ebel (IBS), CEA/CNRS/UJF, Grenoble, FranceSearch for more papers by this authorJohn E. Lunn, John E. Lunn Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam, GermanySearch for more papers by this authorJean-Luc Ferrer, Corresponding Author Jean-Luc Ferrer jean-luc.ferrer@ibs.fr Institut de Biologie Structurale J-P. Ebel (IBS), CEA/CNRS/UJF, Grenoble, FranceInstitut de Biologie Structurale J-P. Ebel (IBS), Laboratoire de Cristallographie et Cristallogenèse des Protéines (LCCP), CEA/CNRS/UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France===Search for more papers by this author Sonia Fieulaine, Sonia Fieulaine Institut de Biologie Structurale J-P. Ebel (IBS), CEA/CNRS/UJF, Grenoble, FranceSearch for more papers by this authorJohn E. Lunn, John E. Lunn Max Planck Institut für Molekulare Pflanzenphysiologie, Potsdam, GermanySearch for more papers by this authorJean-Luc Ferrer, Corresponding Author Jean-Luc Ferrer jean-luc.ferrer@ibs.fr Institut de Biologie Structurale J-P. Ebel (IBS), CEA/CNRS/UJF, Grenoble, FranceInstitut de Biologie Structurale J-P. Ebel (IBS), Laboratoire de Cristallographie et Cristallogenèse des Protéines (LCCP), CEA/CNRS/UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France===Search for more papers by this author First published: 17 May 2007 https://doi.org/10.1002/prot.21481Citations: 8AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Introduction Sucrose-phosphatase (SPP) catalyzes the dephosphorylation of sucrose-6F-phosphate (Suc6P)—the last step in the pathway of sucrose biosynthesis in plants and cyanobacteria.1, 2 SPP from plants contains a catalytic domain that closely resembles the cyanobacterial enzyme.3, 4 Both possess the three conserved motifs that are characteristic of the haloacid dehalogenase (HAD) superfamily of proteins3, 4 and the crystal structure of the Synechocystis sp. PCC 6803 SPP showed that the protein shares a similar fold with other proteins in the HAD superfamily.5 The structure consists of two unequal domains, a larger core domain and a smaller cap domain, both containing a three-layer (αβ)-sandwich.5 The three conserved HAD motifs of SPP form the active site, which is located at the interface between the two domains of the enzyme. The core and cap domains are connected by two hinge loops that allow a closing movement to clasp Suc6P in the active site, bringing the enzyme into its catalytic conformation for the dephosphorylation reaction. The previously reported structures of the Synechocystis SPP also revealed the presence of a glucose-binding pocket within the cap domain, which appears to play a major role in determining the high specificity of the enzyme for its substrate.5 Sucrose is one of the main products of photosynthesis in plants, and suppression of SPP activity in tobacco by RNA interference was reported to lead to chlorosis, inhibition of photosynthesis, and reduced growth rates in plants with less than 10% of wild type activity.6 These observations, and the absence of SPP in animals, suggest that the enzyme is worth considering as a novel herbicide target. Sucrose and several other disaccharides have been reported to inhibit SPP from plants. Maltose and sucrose have been shown to inhibit SPP activity in crude extracts from a wide range of primitive and vascular plants,7 as well as the purified enzyme from pea shoots8 and rice leaves.9 The pea shoot SPP was also reported to be inhibited by melibiose, trehalose, and cellobiose,8 and the rice leaf enzyme is inhibited by turanose, an isomer of sucrose.9 The Synechocystis SPP is also competitively inhibited by sucrose, with an inhibition constant in the millimolar range.4 In this work, we investigated the inhibition of a cyanobacterial SPP enzyme by three glucose-containing disaccharides using a structural approach. The resulting complexes with the Synechocystis SPP confirmed the unique features of the substrate binding site and revealed the structural basis for the competitive inhibition of this enzyme. The results provide a detailed insight into the binding of inhibitory molecules in the active site of SPP, providing a basis for exploring the potential of the enzyme as a novel herbicide target. Materials and methods Crystallization and structure determination Protein expression, purification, and crystallization have been described elsewhere,4, 5 and SPP activity was measured as described in Lunn et al.3 Crystals corresponding to the closed conformation of the enzyme (space group P6522) were incubated for several hours in a 2 μL drop of a solution composed of 3.5M sodium formate, 0.1M Tris-Cl−, pH 8.0, and 33 mM maltose, trehalose, or cellobiose. Cryoprotection was achieved by placing the crystals for about 30 s in a solution composed of 3.0M sodium formate, 20% glycerol, 0.1M Tris-Cl−, pH 8.0, and then the crystals were directly flash frozen in liquid nitrogen using a cryoloop. Data collection was performed at 100 K at the European Synchrotron Radiation Facility (Grenoble, France) on station ID14-eh1, and carried out at λ = 0.934 Å. For each complex, a single crystal was used to collect a complete data set. Date were processed and scaled with the program XDS (Table I).10 Table I. Crystallographic Data and Refinement Statistics Maltose Trehalose Cellobiose X-ray diffraction data Resolution range (Å) 50.0–2.5 50.0–2.2 50.0–2.2 Cell parameters a = b = 68.8, c = 268.8 a = b = 68.9, c = 268.6 a = b = 68.7, c = 268.7 Unique reflections 13,929 20,127 20,122 Completeness (%) 99.9 (99.8) 99.8 (99.2) 99.9 (99.3) I/σ 13.5 (7.6) 12.2 (5.3) 12.2 (5.9) Rsym (%)aa Rsym = ∑|Ii − 〈I〉|/∑Ii, where Ii is the intensity of a reflection and 〈I〉 is the average intensity of that reflection. 17.0 (33.9) 18.5 (45) 19.3 (47.1) Refinement Protein atoms 1,950 1,962 1,950 Solvent molecules 246 300 241 Ligand atoms 23 23 23 Rcryst (%)bb Rcryst = ∑||Fobs| − |Fcalc||/∑|Fobs|. /Rfree (%)cc 5% of the data was set aside for Rfree calculation. 18.4/21.9 17.8/20.0 17.7/19.8 Average B-values for protein (Å2) 16.9 18.3 17.2 Average B-values for ligand (Å2) 19.9 30.9 25.0 RMSD of bonds (Å)/angles (degrees) 0.006/1.3 0.006/1.3 0.006/1.2 Ramachandran plotdd Percentage of residues in most-favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot. 90.2/9.3/0.0/0.5 90.7/8.9/0.0/0.5 91.6/7.9/0.0/0.5 Values in parenthesis are for the outer resolution shell. a Rsym = ∑|Ii − 〈I〉|/∑Ii, where Ii is the intensity of a reflection and 〈I〉 is the average intensity of that reflection. b Rcryst = ∑||Fobs| − |Fcalc||/∑|Fobs|. c 5% of the data was set aside for Rfree calculation. d Percentage of residues in most-favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot. The structures of all SPP complexes were determined using the native model of the protein in its closed conformation (PDB code 1TJ3;5). Each of the structures was refined in a similar manner, with a rigid-body refinement followed by several cycles of positional refinement using CNS11 and manual rebuilding. The first 2FO − FC and FO − FC electron density maps were examined using TURBO-FRODO12 and revealed in each case clear density for both the protein and the ligands. The ligands were built, and water molecules were gradually added during further conjugate gradient refinement with CNS. Water molecules were inserted manually if electron densities of at least 3σ in the difference FO − FC map and 1σ in the 2FO − FC map were located. Statistics for the three refined models are given in Table I. The coordinates and structure factors have been deposited at the RCSB Protein Data Bank with accession codes 2D2V, 2B1Q and 2B1R. Figures were generated using Molscript13 or BOBSCRIPT,14 and rendered using RASTER3D15 or POV-Ray (http://www.povray.org.). Docking experiments Docking experiments were carried out with the GOLD software.16 The search area for the docking was limited to a generous 15 Å-radius sphere around Gly42. Prior to docking, hydrogen atoms were generated for both the protein and the ligands using Chimera.17 Results were evaluated based on the fitness score provided by GOLD and on the root mean squared deviation of the top four hits. Results and discussion SPP-sugar complexes Preliminary experiments confirmed that the Synechocystis SPP is competitively inhibited by sucrose, with a Ki of 161 mM. The enzyme was also found to be inhibited by three glucose-containing disaccharides—maltose (Ki = 100 mM), trehalose (Ki = 26 mM), and cellobiose (Ki = 101 mM)—as well as by glucose (Ki = 354 mM). The enzyme is not inhibited by fructose or galactose. Previously solved SPP crystal structures showed that the enzyme can adopt either an open or a closed conformation,5 but only the closed conformation crystals showed ligand binding and dephosphorylation of Suc6P, indicating that this is the catalytic form of the enzyme. We therefore used SPP crystals in the closed conformation for the soaking experiments with maltose, trehalose, and cellobiose. Each of the three disaccharides was observed in the ligand binding site of the SPP crystals (Fig. 1). The SPP structures presented in this work are similar to the previously solved SPP structures, free or in complex with various ligands,5 the average root mean squared deviation being less than 0.2 Å for 100% of the Cα positions. Figure 1Open in figure viewerPowerPoint The Synechocystis SPP in complex with various ligands. A: Overall structure of the protein in complex with trehalose. β-strands and α-helices are in green and purple, respectively. Trehalose is drawn in ball-and-stick format. B: maltose, C: trehalose, and D: cellobiose in ball-and-stick format are shown in their FO − FC electron density omit map contoured at 2σ. As observed in the structures of SPP with Suc6P or sucrose bound in the active site,5 maltose, trehalose, and cellobiose are held in the ligand binding site by interactions between oxygen atoms (O3, O4, and O6) from one of their glucose rings and residues from the cap domain of SPP (Gln107, Lys116, and Asn189) (Fig. 2). An additional interaction is observed in the SPP-trehalose complex, between atom O2′ from trehalose and atom NZ from Lys152 of the enzyme (Fig. 2), this interaction being also observed in the previously solved SPP-sucrose complex.5 The three glucose-containing disaccharides thus share a similar mode of binding to SPP with the natural ligands of the enzyme (Suc6P and sucrose), providing a simple explanation for the competitive nature of the inhibition of SPP by these sugars. Figure 2Open in figure viewerPowerPoint The ligand binding site of the Synechocystis SPP in complex with trehalose. Residues that are involved in the trehalose binding are shown in ball-and-stick format. Carbon, nitrogen, oxygen, and phosphorus atoms are colored in black, blue, red, and pink, respectively. A noncatalytic magnesium ion (ncMg, shown as a grey sphere) is observed in the active site. Dashed lines indicate the hydrogen bonds and the salt bridges. A unique substrate binding site Synechocystis SPP belongs to the HAD superfamily and has particularly high structural homology with a phosphoglycolate phosphatase from Thermoplasma acidophilum and a phosphorylated-carbohydrate phosphatase from Thermotoga maritima.5 The three SPP structures solved in this work were used to run a new search for structural homology, using DALI.18 Two recently solved structures of bacterial proteins were found, both of which are sugar-phosphatases. The first of these, the BT4131 protein from Bacteroides thetaiotaomicron VPI-5482 (PDB accession 1YMQ), appears to be a broad-range sugar phosphatase, with fructose 6-phosphate or a close structural analog being likely substrates in vivo.19 The second structural homologue of SPP is the YbiV protein from Escherichia coli K12 (PDB accession 1RLM). Although this protein can also hydrolyze several different types of sugar-phosphates, of which ribose 5-phosphate was the preferred substrate out of the compounds tested, the natural substrate of the enzyme was not identified.20 When these two structures are superimposed on that of the SPP-maltose complex, 180 residues occupy identical positions within a maximum of 2.4 Å Cα root mean squared deviation, although amino acid sequence identity is only 18%. As expected, the catalytic centers in the active sites of SPP and these two related proteins are very similar, and include the three conserved signature motifs of the HAD superfamily. However, there are significant differences between the three enzymes in other regions of the active site that are involved in substrate binding. As previously discussed, Suc6P is held in the substrate binding site of SPP by interactions with its glucose ring. However, steric hindrance in the substrate binding sites of the BT4131 and YbiV proteins prevents binding of sugar-phosphate molecules with more than one sugar ring, such as Suc6P or trehalose-6-phosphate.19, 20 Another significant difference is that the fructose ring of Suc6P does not interact with SPP, whereas in the other two phosphatases the glycone part of the substrate is held in the substrate binding site by aromatic residues that form a hydrophobic cavity. These aromatic residues, which are a common feature of carbohydrate binding proteins, allow a stacking interaction with the ligand.21-23 We also compared the SPP-disaccharide complexes with publicly available structures of other proteins complexed with maltose, trehalose, or cellobiose, but found no obvious similarities. This observation, together with the previously noted lack of similarity of SPP to other sucrose-binding proteins,5 confirms that the substrate-binding site of SPP represents a novel type of disaccharide-binding domain. Concepts for design of novel inhibitors of SPP The unique features of the substrate-binding site of the SPP enzyme should allow design of specific inhibitors that do not inhibit other HAD superfamily enzymes or sugar-binding proteins. The critical role of the glucose-binding site in substrate specificity suggested that molecules with a glucose ring able to mimic the glucose moiety of Suc6P have good potential to inhibit SPP. The second critical feature for Suc6P binding in the active site of SPP is the phosphate group.5 This could be replaced by a phosphonate group, which would be expected to mimic the phosphate group of Suc6P, but would be resistant to hydrolysis. Finally, the glucose and phosphonate groups need to be joined by some kind of linker to maintain an appropriate distance between the two ends of the molecule. An in silico docking approach with the GOLD software16 was used to test these concepts for design of novel inhibitors of SPP. The best hits were graphically and numerically compared with the binding mode of Suc6P, the natural substrate of the enzyme, in the structure of the SPP-Suc6P complex5 (data not shown). From this analysis, the most effective inhibitor was predicted to be the molecule shown in Figure 3(B), which contains a glucose ring, as in Suc6P, and a pseudo-fructose ring attached to an α-keto phosphonate group. Figure 3Open in figure viewerPowerPoint Chemical structures of potential inhibitors of SPP. A: Suc6P, the substrate. B: The best virtual molecule obtained by in silico design and tested by docking simulations. C: 4-Nitrophenyl-α-L-galactopyranoside, the best molecule out of a library of over 300 commercially available compounds containing a hexose moiety, tested by docking simulations. In parallel with the rational design approach, we also performed a massive docking simulation on a library of over 300 compounds containing a hexose moiety (Ref.24; http://blaster.docking.org/zinc/). Most of the compounds were predicted to bind poorly to SPP, and the molecule with the best fitness score was 4-nitrophenyl-α-L-galactopyranoside (Zinc code: 156947; C.A.S. No 10357-27-4) [Fig. 3(C)]. The effect of this compound on Synechocystis SPP activity was tested experimentally. Measurements showed up to 50% inhibition of SPP activity in the presence of 80 mM 4-nitrophenyl-α-L-galactopyranoside, but this inhibition does not appear to be competitive, as the Vmax was decreased in the presence of inhibitor. Inhibition of SPP by sugars and the control of sucrose synthesis in cyanobacteria Sucrose and trehalose are used as compatible solutes by some cyanobacteria when exposed to osmotic stress. The inhibition of SPP by sucrose will limit the amount of sucrose accumulated, and the even greater sensitivity of the enzyme to trehalose could explain why trehalose-synthesizing cyanobacteria usually have very little sucrose. Interestingly, Synechocystis sp. PCC6803 synthesizes very little sucrose and no trehalose, but accumulates the glucose-containing heteroside, glucosylglycerol, instead.25 From our detailed understanding of the active site of SPP, we can confidently predict that this molecule should also inhibit the enzyme, and that such inhibition probably restricts the synthesis of sucrose in this species. Finally, preliminary in silico docking experiments showed that compounds containing a glucose-like ring, linked to an α-keto phosphonate group via a fructose-like ring, should be potent inhibitors of the cyanobacterial SPP. This result provides encouragement for use of this approach to discover novel inhibitors of plant enzymes that are potential herbicide targets. This approach might also be used to discover potential ligands for the many HAD superfamily proteins of unknown function, to help identify the physiological substrates of these enzymes, and reveal their function.26 Acknowledgements We thank the staff of ESRF in Grenoble for making station ID14-eh1 available. We are also grateful to Laurent Griffe (CNRS, France) and Thomas J. Baiga (The Salk Institute, CA, USA) for their assistance in the design of inhibitors. REFERENCES 1 Leloir LF, Cardini CE. The biosynthesis of sucrose phosphate. J Biol Chem 1955; 214: 157– 165. CASPubMedGoogle Scholar 2 Hawker JS, Hatch MD. A specific sucrose phosphatase from plant tissues. Biochem J 1966; 99: 102– 107. CrossrefCASPubMedWeb of Science®Google Scholar 3 Lunn JE, Ashton AR, Hatch MD, Heldt HW. Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants. Proc Natl Acad Sci USA 2000; 97: 12914– 12919. CrossrefCASPubMedWeb of Science®Google Scholar 4 Lunn JE. Evolution of sucrose synthesis. Plant Physiol 2002; 128: 1490– 1500. CrossrefCASPubMedWeb of Science®Google Scholar 5 Fieulaine S, Lunn JE, Borel F, Ferrer JL. The structure of a cyanobacterial sucrose-phosphatase reveals the sugar tongs that release free sucrose in the cell. Plant Cell 2005; 17: 2049– 2058. CrossrefCASPubMedWeb of Science®Google Scholar 6 Chen S, Hajirezaei M, Peisker M, Tschiersch H, Sonnewald U, Börnke F. Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth. Planta 2005; 221: 479– 492. CrossrefCASPubMedWeb of Science®Google Scholar 7 Hawker JS, Smith GM. Occurrence of sucrose phosphatase in vascular and non-vascular plants. Phytochemistry 1984; 23: 245– 249. CrossrefCASWeb of Science®Google Scholar 8 Whitaker DP. Purification and properties of sucrose-6-phosphatase from Pisum sativum shoots. Phytochemistry 1984; 23: 2429– 2430. CrossrefCASPubMedWeb of Science®Google Scholar 9 Echeverria E, Salerno G. Properties of sucrose-phosphate phosphatase from rice (Oryza sativa) leaves. Plant Sci 1994; 96: 15– 19. CrossrefCASWeb of Science®Google Scholar 10 Kabsch W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Crystallogr 1993; 26: 795– 800. Wiley Online LibraryCASWeb of Science®Google Scholar 11 Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr Sect D. Biol Crystallogr 1998; 54 (Pt 5): 905– 921. Wiley Online LibraryPubMedGoogle Scholar 12 Roussel A, Cambillau C. TURBO-FRODO. In: Silicon graphics geometry partners directory. Mountain View, CA: Silicon Graphics; 1989, pp. 77– 78. Web of Science®Google Scholar 13 Esnouf RM. An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. J Mol Graph Model 1997; 15: 112– 134. CrossrefWeb of Science®Google Scholar 14 Esnouf RM. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr Sect D. Biol Crystallogr 1999; 55 (Pt 4): 938– 940. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 15 Merritt EA, Bacon DJ. Raster3D: photorealistic molecular graphics. Methods Enzymol 1997; 277: 505– 524. CrossrefCASPubMedWeb of Science®Google Scholar 16 Jones G, Willett P, Glen RC. Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. J Mol Biol 1995; 245: 43– 53. CrossrefCASPubMedWeb of Science®Google Scholar 17 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem 2004; 25: 1605– 1612. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 18 Holm L, Sander C. Protein structure comparison by alignment of distance matrices. J Mol Biol 1993; 233: 123– 138. CrossrefCASPubMedWeb of Science®Google Scholar 19 Lu Z, Dunaway-Mariano D, Allen KN. HAD superfamily phosphotransferase substrate diversification: structure and function analysis of HAD subclass IIB sugar phosphatase BT4131. Biochemistry 2005; 44: 8684– 8696. CrossrefCASPubMedWeb of Science®Google Scholar 20 Roberts A, Lee SY, McCullagh E, Silversmith RE, Wemmer DE. YbiV from Escherichia coli K12 is a HAD phosphatase. Proteins 2005; 58: 790– 801. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 21 Vyas NK, Vyas MN, Quiocho FA. Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein. Science 1988; 242: 1290– 1295. CrossrefCASPubMedWeb of Science®Google Scholar 22 Vyas NK. Atomic features of protein–carbohydrate interactions. Curr Opin Struct Biol 1991; 1: 732– 740. CrossrefCASWeb of Science®Google Scholar 23 Duan X, Hall JA, Nikaido H, Quiocho FA. Crystal structures of the maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding. J Mol Biol 2001; 306: 1115– 1126. CrossrefCASPubMedWeb of Science®Google Scholar 24 Irwin JJ, Shoichet BK. ZINC—a free database of commercially available compounds for virtual screening. J Chem Inf Model 2005; 45: 177– 182. CrossrefCASPubMedWeb of Science®Google Scholar 25 Lunn JE, Price GD, Furbank RT. Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 1999; 40: 297– 305. CrossrefCASPubMedWeb of Science®Google Scholar 26 Burroughs AM, Allen KN, Dunaway-Mariano D, Aravind L. Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes. J Mol Biol 2006; 361: 1003– 1034. CrossrefGoogle Scholar Citing Literature Volume68, Issue315 August 2007Pages 796-801 FiguresReferencesRelatedInformation
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