Probing the Mechanism of Ligand Recognition in Family 29 Carbohydrate-binding Modules
2005; Elsevier BV; Volume: 280; Issue: 25 Linguagem: Inglês
10.1074/jbc.m501551200
ISSN1083-351X
AutoresJ.E. Flint, David N. Bolam, Didier Nurizzo, Edward J. Taylor, Michael P. Williamson, Christopher R. Walters, G.J. Davies, Harry J. Gilbert,
Tópico(s)Plant nutrient uptake and metabolism
ResumoThe recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 Å resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both β-manno- and β-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity. The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 Å resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both β-manno- and β-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity. IntroductionThe plant cell wall comprises the most abundant source of organic carbon on the planet. This extensive resource is made available to the biosphere through the action of microbial glycoside hydrolases, which are thus of considerable biological and industrial importance. Plant cells are surrounded by an intimate network of polysaccharides that are highly inaccessible to enzyme attack (1Brett C.T. Waldron K. Physiology and Biochemistry of Plant Cell Walls. Chapman and Hall, London1996: 4-64Google Scholar). Glycoside hydrolases that attack the plant cell wall frequently display a modular structure in which non-catalytic carbohydrate-binding modules (CBMs) 1The abbreviations used are: CBM, carbohydrate-binding module; AGE, affinity gel electrophoresis; BSA, bovine serum albumin; CGM, Carob galactomannan; HEC, hydroxyethyl cellulose; hvKGM, Konjac high viscosity glucomannan; ITC, isothermal titration calorimetry; lvKGM, Konjac low viscosity glucomannan. target the biocatalysts to specific polysaccharides (for review, see Ref. 2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1488) Google Scholar). By bringing the enzymes into intimate and prolonged contact with their recalcitrant substrate, CBMs potentiate the activity of glycoside hydrolases against the plant cell wall and therefore play a pivotal role in the saccharification of plant tissue by these enzymes (3Bolam D.N. Ciruela A. McQueen-Mason S. Simpson P. Williamson M.P. Rixon J.E. Boraston A. Hazlewood G.P. Gilbert H.J. Biochem. J. 1998; 331: 775-781Crossref PubMed Scopus (236) Google Scholar, 4Gill J. Rixon J.E. Bolam D.N. McQueen-Mason S. Simpson P.J. Williamson M.P. Hazlewood G.P. Gilbert H.J. Biochem. J. 1999; 342: 473-480Crossref PubMed Scopus (69) Google Scholar).CBMs are grouped into 42 sequence-based families (5Coutinho P.M. Henrissat B. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Engineering. Royal Society of Chemistry, Cambridge, UK1999: 3-12Google Scholar), which may be found in the continuously updated carbohydrate-active enzyme data base at afmb.cnrs-mrs.fr/CAZY/. Virtually all CBMs characterized to date are β-stranded proteins arranged in what some term a "jelly-roll" motif. CBMs have also been grouped into three types based on the topology of the binding sites, which reflects the macromolecular structure of the target ligand (2Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1488) Google Scholar, 6Boraston A.B. McLean B.W. Kormos J. Alamm M.M. Gilkes N.R. Haynes C.A. Tomme P. Kilburn D.G. Warren R.A.J. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Engineering. Royal Society of Chemistry, Cambridge, UK1999: 202-211Google Scholar). 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There is, therefore, considerable variation in ligand specificity within specific CBM Type B families (for example, Refs. 12Boraston A.B. Nurizzo D. Notenboom V. Ducros V. Rose D.R. Kilburn D.G. Davies G.J. J. Mol. Biol. 2002; 319: 1143-1156Crossref PubMed Scopus (126) Google Scholar and 21Abou Hachem M. Nordberg Karlsson E. Bartonek-Roxa E. Raghothama S. Simpson P.J. Gilbert H.J. Williamson M.P. Holst O. Biochem. J. 2000; 345: 53-60Crossref PubMed Scopus (95) Google Scholar).A number of factors contribute to carbohydrate binding and recognition by CBMs. Regardless of specificity, there is substantial, and growing, evidence that the stacking of aromatic residues against the pyranose rings of polysaccharides plays a critical role for both Type A and Type B CBMs (16Czjzek M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. 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Protein Eng. 2000; 13: 801-809Crossref PubMed Google Scholar). In Type B modules, however, binding is enthalpically driven, and mutagenesis studies suggest that direct hydrogen bonds play an important role in saccharide binding (for example, Refs. 16Czjzek M. Bolam D.N. Mosbah A. Allouch J. Fontes C.M. Ferreira L.M. Bornet O. Zamboni V. Darbon H. Smith N.L. Black G.W. Henrissat B. Gilbert H.J. J. Biol. Chem. 2001; 276: 48580-48587Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 27Xie H. Gilbert H.J. Charnock S.J. Davies G.J. Williamson M.P. Simpson P.J. Raghothama S. Fontes C.M. Dias F.M. Ferreira L.M. Bolam D.N. Biochemistry. 2001; 40: 9167-9176Crossref PubMed Scopus (78) Google Scholar, and 30Henshaw J.L. Bolam D.N. Pires V.M. Czjzek M. Henrissat B. Ferreira L.M. Fontes C.M. Gilbert H.J. J. Biol. Chem. 2004; 279: 21552-21559Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although, in some cases indirect (water-mediated) hydrogen bonds appear to play a role in the binding of lectins to their ligands (31Ravishankar R. Ravindran M. Suguna K. Surolia A. Vijayan M. Curr. Sci. India. 1997; 72: 855-861Google Scholar, 32Ravishankar R. Suguna K. Surolia A. Vijayan M. Acta Crystallogr. Sect. D. 1999; 55: 1375-1382Crossref PubMed Scopus (57) Google Scholar, 33Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (468) Google Scholar), the importance of these solvent-mediated interactions in polysaccharide recognition by CBMs is unclear.Family 29 contains just two members, termed CBM29-1 and CBM29-2, which are components of the Piromyces equi non-catalytic protein NCP1 (34Freelove A.C. Bolam D.N. White P. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 2001; 276: 43010-43017Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). NCP1 is present in the large, highly efficient, extracellular multienzyme plant cell wall-degrading complex produced by this anaerobic fungus. Both of the CBM29 modules bind to a range of β-1,4-linked polysaccharides that include cellulose, xylan, mannan, and glucomannan, although CBM29-1 displays lower affinity for these ligands than CBM29-2. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components of the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote. We described, previously, the crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose (15Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (82) Google Scholar). Although these structures revealed an open ligand binding cleft providing six sugar binding subsites, they left unanswered questions concerning the structural basis for the difference in affinity between the two family 29 modules and the functional importance of direct and indirect interactions between CBM29-2 and its ligands, particularly with respect to the importance of O2 (which is axial in mannose and equatorial in glucose) as a specificity determinant.Here we report the crystal structure of CBM29-1 and, by comparison with CBM29-2, provide insight into the structural basis for the different affinities displayed by the two family 29 modules. A mutagenesis approach to interrogate the functional importance of direct and indirect interactions between CBM29-2 and its ligands shows that hydrophobic stacking between aromatic residues and sugar rings dominates the binding affinity, as seen for other Type A and Type B CBMs. Although direct hydrogen bonds between specific amino acids in CBM29-2 and the hydroxyl groups of the sugar ligands make a significant contribution to overall affinity, the interactions between the protein and either the axial or equatorial O2 of mannose and glucose, respectively, contribute little to the overall binding energy. In contrast with studies on some lectins, we show that water-mediated hydrogen bonds contribute very little to ligand recognition by CBM29.EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Culture ConditionsThe Escherichia coli strains used in this study were BL21(DE3): pLysS and Origami B:pLysS (Novagen). The plasmids employed in this work were recombinants of the expression vector pET22b (Novagen) encoding CBM29-2 (pVM1), and CBM29-1 (pVM2), which were constructed previously (15Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (82) Google Scholar, 34Freelove A.C. Bolam D.N. White P. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 2001; 276: 43010-43017Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). CBM29-1 and CBM29-2, which comprise residues 174–317 and 336–479, respectively of P. equi NCP1, each contain a C-terminal His6 tag (15Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (82) Google Scholar). E. coli was cultured in Luria-Bertoni broth liquid media at 37 °C and 180 rpm unless otherwise stated. When culturing strains that harbor the pET-based recombinant plasmids, media were supplemented with 50 μg/ml ampicillin.Generation of Mutants of CBM29-1 and CBM29-2Derivatives of the two CBMs were generated by the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using pVM1 and pVM2 as template DNA. The primers employed in the mutagenesis PCRs are shown in Table 1S. The complete sequences of the DNA encoding the CBM29 mutants were determined by MWG-Biotech (Ebersberg, Germany) using T7 forward and reverse primers to confirm that only the desired mutations had been introduced.Expression and Purification of CBM29-1 and CBM29-2CBM29-2 was expressed in E. coli BL21(DE3):pLysS as described previously (15Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (82) Google Scholar, 34Freelove A.C. Bolam D.N. White P. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 2001; 276: 43010-43017Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar); CBM29-1 was produced in E. coli Origami B:pLysS. Briefly, cells were cultured in 1 liter of Luria-Bertoni broth in 2-liter baffled flasks at 30 °C and 180 rpm to an A600 ~0.6. Recombinant protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 200 μm and incubation at 16 °C for 16 h. CBM29-2 was purified by immobilized metal ion affinity chromatography using Talon resin (Clontech), anion exchange, and size exclusion chromatography as described by Charnock et al. (15Charnock S.J. Bolam D.N. Nurizzo D. Szabo L. McKie V.A. Gilbert H.J. Davies G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14077-14082Crossref PubMed Scopus (82) Google Scholar). CBM29-1 was purified using the same procedure as for CBM29-2 except that the protein was eluted from the Talon resin with 5 mm rather than 100 mm imidazole. The CBMs were all purified to electrophoretic homogeneity as judged by SDS-PAGE. The protein concentration was determined from the calculated molar extinction coefficient of CBM29-1 and CBM29-2 at 280 nm, which were 26,860 and 26,150 m–1 cm–1, respectively, and reduced coefficients of 20,340 and 25,560 m–1 cm–1 were used for the CBM29-2 tryptophan and tyrosine mutants, respectively.Ligand Binding StudiesLigand binding was determined by both affinity gel electrophoresis (AGE) and isothermal titration calorimetry (ITC). AGE was performed as described previously (34Freelove A.C. Bolam D.N. White P. Hazlewood G.P. Gilbert H.J. J. Biol. Chem. 2001; 276: 43010-43017Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) using Konjac high viscosity glucomannan (hvKGM), Carob galactomannan (CGM), and hydroxyethyl cellulose (HEC), which were purchased from Megazyme International (Bray, County Wicklow, Ireland). The polysaccharides were included in the gels at concentrations ranging from 0.125 to 10 mg/ml. Kd values were calculated by determining the mobility of the CBM, in the presence and absence of ligand, relative to a nonbinding standard BSA (Sigma) as originally described by Takeo (35Takeo K. Electrophoresis. 1984; 5: 187-193Crossref Scopus (73) Google Scholar). ITC measurements were made at 25 and 10 °C following standard procedures (36Flint J. Nurizzo D. Harding S.E. Longman E. Davies G.J. Gilbert H.J. Bolam D.N. J. Mol. Biol. 2004; 337: 417-426Crossref PubMed Scopus (35) Google Scholar) using a Microcal Omega titration calorimeter. Proteins were dialyzed, extensively, against 50 mm sodium phosphate buffer, pH 7.0, and the ligand was dissolved in the same buffer to minimize heats of dilution. During a titration experiment the protein sample (100–800 μm), stirred at 300 rpm in a 1.4331-ml reaction cell maintained at 25 or 10 °C, was injected with 25 successive 10-μl aliquots of ligand comprising 20 mg/ml low viscosity KGM (lvKGM) at 200-s intervals. The molar concentration of CBM29-2 binding sites present in the glucomannan was determined as described previously (37Szabo L. Jamal S. Xie H. Charnock S.J. Bolam D.N. Gilbert H.J. Davies G.J. J. Biol. Chem. 2001; 276: 49061-49065Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Integrated heat effects, after correction for heats of dilution, were analyzed by nonlinear regression using a single site binding model (Microcal Origin, version 5.0). The fitted data yield the association constant (Ka) and the enthalpy of binding (ΔH). Other thermodynamic parameters were calculated using the standard thermodynamic equation. −RTlnKa=ΔG=ΔH−TΔS(Eq. 1) NMR SpectroscopyTo assess the structural integrity of the mutant proteins, one-dimensional NMR analysis was performed on immobilized metal ion affinity chromatography-purified wild type and mutant forms of CBM29-2. Protein samples were ~500 μm in 10 mm sodium phosphate buffer, pH 6.5, containing 10% D2O. NMR spectra were recorded at 30 °C on a Bruker DRX-500 spectrometer, and 1H chemical shifts were referenced to an internal standard of 3-(trimethylsilyl)propionate-2,2,3,3-d4 at 0.00 ppm. Data were processed by FELIX (Accelrys, Inc., San Diego).Crystallization of CBM29Wild type CBM29-1 and CBM29-2 mutants were screened for crystallization with the Clear Strategy Screen (38Brzozowski A.M. Walton J. J. Appl. Crystallogr. 2001; 34: 97-101Crossref Scopus (72) Google Scholar) at 20 °C with a protein concentration of 10–20 mg/ml. Tetragonal bipyramidal crystals of uncomplexed CBM29-2 mutants were obtained after 2 days from drops that comprised 2 μl of the CBM29-2 mutants Y46A, R112A, or D114A at 10 mg/ml and 1 μl of 100 mm Na/HEPES buffer, pH 7.5, containing 150 mm KSCN, 20% (v/v) ethylene glycol, and 16–21% (w/v) polyethylene glycol 3350. CBM29-2 D83A in complex with mannohexaose was crystallized at 10 mg/ml in 1.5 m ammonium sulfate, 0.1 m sodium citrate, pH 6.5, and with 10 mm mannohexaose (25% glycerol was subsequently included in the stabilizing solution as a cryoprotectant). The CBM29-2 K74A, with cellohexaose, was crystallized in 0.18 m ammonium tartrate, 18% polyethylene glycol 3350, 25% 2-methyl-2,4-pentanediol, 5% glycerol, 10 mm cellohexaose (and the same liquor used as a cryoprotectant). Unliganded CBM29-1 was crystallized at 10 mg/ml in 0.6 m lithium acetate 20% polyethylene glycol 3350, pH 7.8, and grown at 20 °C for 6 weeks producing amorphous crystals. Freshly prepared drops were then streak-seeded from crushed amorphous crystals to yield more regular crystals after 2 weeks. Crystals were then harvested into a cryoprotectant mother liquor containing an additional 25% glycerol. Crystals of CBM29-1 in the presence of 5 mm cellohexaose were obtained, after 4 weeks, in drops comprising 10 mg/ml protein in 0.2 m lithium chloride, 20% polyethylene glycol 3350, pH 6.7. 25% glycerol was added to the mother liquor for subsequent freezing.Data Collection and Structure ResolutionCBM29-2 Mutants—Data were collected, to between 2.25 and 1.6 Å resolution, on beamline ID14-EH1 at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) and beamline PX9.6 at the Daresbury (UK) Synchrotron Radiation Source. Crystals all belong to the orthorhombic space group P43212. Crystal data and final refinement statistics are reported in Table I. Data were processed and scaled using MOSFLM and SCALA from the CCP4 suite (39Project Collaborative Computational Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar) or DENZO and SCALEPACK from the HKL suite (40Otwinowski Z. Minor W. Macromol. Crystallogr. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). Structures were solved by molecular replacement using the program AMoRe (41Navaza J. Saludijan P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar) using the wild type CBM29-2 structure, in the absence of ligand (1GWK), as the search model. After molecular replacement, maximum likelihood-based refinement of the atomic positions and temperature factors were performed with REFMAC (42Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar). This procedure was interspersed with manual correction using the X-AUTOFIT option within QUANTA (Accelrys, Inc.). Water molecules were first placed automatically with REFMAC/ARP (42Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar, 43Lamzin V.S. Wilson K.S. Acta Crystallogr. Sect. D. 1993; 49: 129-147Crossref PubMed Google Scholar) and verified manually.Table IData and structure quality statistics for the CBM29-2 mutantsProteinY46AK85AR112AD114AD83AK74ARadiation sourceID14-EH1ID14-EH1PX9.6ID14-EH1PX9.6ID14-EH1Nb images200270184High 270360High 270Low 135Low 135DetectorADSC quantum 4ADSC quantum 4ADSC quantum 4ADSC quantum 4ADSC quantum 4ADSC quantum 4SoftwareMOSFLM/SCALAMOSFLM/SCALAMOSFLM/SCALAMOSFLM/SCALADENZO/SCALEPACKDENZO/SCALEPACKSpace groupP43 21 2P43 21 2P43 21 2P43 21 2C2C 2Cell (Å, °)a = b = 92.5, c = 80.8a = b = 93.0, c = 83.2a = b = 91.8, c = 79.9a = b = 93.2, c = 82.3a = 51.2, b = 42.6, c = 60.2, β = 93.7a = 107.7, b = 42.8, c = 35.3, β = 105.2Wavelength (Å)0.9340.9340.870.9340.870.934Resolution (Å)40.0-2.10 (2.21-2.10)38.0-1.85 (1.95-1.85)26.0-2.2538.0-1.6 (1.71-1.60)100.0-1.3 (1.32-1.30)20.0-1.4 (1.45-1.40)Unique reflections21,01931,74815,51448,33831,89230,504Completeness (%)99.9 (100)100 (100)92.8 (70.6)100.0 (100.0)99.8 (98.1)99.4 (99.9)Rsym (%)8.1 (41.2)8.5 (66.5)5.8 (56.7)6.7 (70.7)5.5 (23.1)5.1 (10.2)Multiplicity7.3 (7.5)10.5 (10.6)5.8 (3.0)11.9 (7.4)3.6 (2.7)3.7 (3.7)I/σI21.8 (4.3)22.1 (4.1)23.1(1.6)22.8 (2.0)32.4 (6.6)30.0 (12.2)RefinementRcryst (%)20.017.320.418.616.118.6Rfree (%)23.421.125.420.917.520.9r.m.s.ar.m.s., root mean square. on bond distances (Å)0.0170.0170.0180.0180.0180.018r.m.s. on bond angles (°)1.6061.5741.6971.5531.9081.553PDB code1W8W1W8Z1W9F1W901W8U1W8Ta r.m.s., root mean square. Open table in a new tab CBM29-1—Data for the unliganded CBM29-1 were collected on a single crystal at 100 K, on beamline ID14-4 of the ESRF. Data were integrated, scaled, and reduced with the HKL suite (40Otwinowski Z. Minor W. Macromol. Crystallogr. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). The crystal belongs to space group P212121 with unit cell dimensions a = 30.1, b = 57.5, c = 76.1 Å. Data extend to 1.5 Å resolution (outer shell 1.53–1.5 Å) with Rmerge: 3.8% (9.5%), a multiplicity of 5 (3.2) observations/reflection, and mean I/σI of 40 (13Carvalho A.L. Goyal A. Prates J.A. Bolam D.N. Gilbert H.J. Pires V.M. Ferreira L.M. Planas A. Romao M.J. Fontes C.M. J. Biol. Chem. 2004; 279: 34785-34793Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The data are 100% (99%) complete. Molecular replacement was attempted, using the three-dimensional structure of CBM29-2 (1GWK) as the search model but was unsuccessful.Data for CBM29-1 in complex with cellohexaose crystals were collected to 2.05 Å (outer shell 12.12–2.05 Å) on beamline ID14-EH2 of the ESRF and processed with the HKL suite. Data have Rmerge: 6% (49%), a multiplicity of 3.6 (2.6) observations/reflection, and mean I/σI of 25 (3.1). The data are 99% (91%) complete. The crystal belongs to space group P21, with cell dimensions a = 40.1, b = 60.1, c = 62.9 Å, and β = 108.3° and has two similarly orientated molecules in the asymmetric unit related by a translation-only vector of 0.5, 0, 0.5. Molecular replacement in this form, using the three-dimensional structure of CBM29-2 (1GWK) as the search model and the program AMoRe (41Navaza J. Saludijan P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar) DUPS with default settings and data to 3.0 Å resolution, was successful.After molecular replacement, maximum likelihood-based refinement of the atomic positions and temperature factors was performed with REFMAC (42Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar). At this point the partially refined model was used for molecular replacement of the original uncomplexed form (above) whose data were superior and the CBM29-1 structure refined with REFMAC with manual correction using the X-AUTOFIT option within QUANTA (Accelrys, Inc.). Water molecules were first placed automatically with ARP/REFMAC (42Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Sc
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