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• W1983125435 abstract "Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-β-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc (β-d-galactosyl-1,4-β-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Å, respectively. Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-β-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc (β-d-galactosyl-1,4-β-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Å, respectively. Clostridium perfringens is a Gram-positive, spore-forming, non-motile, rod-shaped anaerobe. As a pathogen of humans, C. perfringens is often associated with gas gangrene, necrotic enteritis, and, most commonly, food poisoning (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar, 3Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar). Determination of the genome sequence of C. perfringens (strain 13) (4Shimizu T. Ohshima S. Ohtani K. Hayashi H. Microbiol. Immunol. 2001; 45: 179-189Crossref PubMed Scopus (22) Google Scholar) has revealed at least 54 open reading frames encoding putative glycoside hydrolases falling into 24 known glycoside hydrolase families (see afmb.cnrs-mrs.fr/CAZY/index.html) (5Coutinho P.M. Henrissat B. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge1999: 3-12Google Scholar). Many encode intracellular proteins likely involved in the latter stages of sugar metabolism or proteins involved in peptidoglycan remodeling; however, roughly one-half are predicted to be secreted and are likely involved in the early stages of sugar metabolism. Although C. perfringens is most frequently thought of as a “flesh-eater,” its most common niche in humans is the gastrointestinal tract. However, very few, if any, of the secreted glycoside hydrolases have predicted substrate specificities consistent with metabolism of dietary polysaccharides in the human gut making gastric mucins, highly hydrated glycoproteins comprising up to 80% carbohydrate, the most likely target of the secreted C. perfringens enzymes. Indeed, the majority of these enzymes are predicted to have specificities appropriate for the degradation of complex glycans, suggesting that this bacterium is well equipped to attack the diverse sugar structures of the mucins in this environment. Consistent with this is the mucosal necrosis associated with severe enteritis caused by C. perfringens (6Gerding D.N. Johnson S. Rood J.I. McClane B.A. Songer J.G. Titball R.W. The Clostridia Molecular Biology and Pathogenesis. Harcourt Brace & Company, London1997: 117-140Google Scholar), which may be in part due to the arsenal of C. perfringens glycoside hydrolases. In turn, breaking down the mucosal barrier could improve access of other toxins, such as the pore-forming cpe (C. perfringens enterotoxin), to the epithelial layer. Thirteen of the predicted C. perfringens (strain 13) glycoside hydrolases (and notably 13 glycoside hydrolases for each of the sequenced Bacteroides sp. genomes (thetaiotaomicron, fragilis YCH46, and fragilis 25285)) are highly modular and have, in addition to catalytic domains, modules with amino acid sequence identity to family 32 carbohydrate-binding modules (CBMs) 3The abbreviations used are: CBM, carbohydrate-binding module; ITC, isothermal titration calorimetry; r.m.s.d., root mean square deviation. (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar). CBMs are generally considered to be modules with carbohydrate-binding function, but no catalytic activity, that are found within the modular architectures of glycoside hydrolases (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). They are currently classified into 45 families based on amino acid sequence identity (see afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.) and loosely grouped into three types, A (crystalline polysaccharide binding), B (polysaccharide chain binding), and C (small sugar binding or “lectin-like”), established by functional properties (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). Based on limited biochemical evidence, the family 32 CBMs appear to be type C CBMs. The x-ray crystal structures of the Cladobotryum dendroides galactose oxidase and the Micromonospora viridifaciens sialidase (MvGH33) revealed the β-sandwich lectin-like folds of their cognate CBM32 modules (9Ito N. Phillips S.E. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D. Knowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (697) Google Scholar, 10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Co-crystallizations of MvGH33 with galactose showed the potential of its CBM32 module, here called MvCBM32, to bind galactose (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Newstead S.L. Watson J.N. Bennet A.J. Taylor G. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 1483-1491Crossref PubMed Scopus (43) Google Scholar), which was subsequently verified by functional studies (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar). Thus, the family 32 CBMs, which are often referred to as F5/8 domains, have been generally considered as galactose binding domains. The family 32 CBMs stand out among the CBM families, because they are frequently found appended to enzymes with “exotic” specificities (e.g. sialidases, β-hexosaminidases, mannosidases, and fucosidases) and are found in bacteria capable of causing disease in humans. In contrast, the vast majority of CBMs in other families are found appended to enzymes that are active on plant cell wall polysaccharides. In the context of the plant cell wall hydrolases, the function of CBMs has been repeatedly shown to be to localize the enzyme to an appropriate substrate (8Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). By analogy to the plant cell wall hydrolases, the role of family 32 CBMs is likely to target their parent enzymes to carbohydrate substrates; however, with these CBMs the substrates are likely more complex glycans, such as gastric mucins in the case of C. perfringens and Bacteroides sp. Previous studies of family 32 CBMs have not addressed the possibility that the specificity of these CBMs extend beyond a preference simply for galactose and may actually include specificity for complex glycan chains. Thus, studies of family 32 CBMs from bacterial pathogens enter a new area of carbohydrate-binding module-mediated host-pathogen interactions and will extend our knowledge of this potentially complex family of carbohydrate-binding proteins. To better understand CBM32 structure and function we initiated studies of CpGH84C from C. perfringens (strain ATCC 13124). This enzyme, which comprises four modules defined on the basis of primary structure comparisons (see Fig. 1), was chosen as a model system, because, relative to other family 32 CBM-containing enzymes, it is reasonably small and has a simple modular architecture that is amenable to accurate definition of the modular boundaries. To facilitate structure-function studies, we dissected this protein at the genetic level to recombinantly produce isolated CpCBM32. The experimental results reveal the ability of the CBM to bind to terminal glycotopes commonly found in elaborated O- and complex N-glycans (12Gupta D. Kaltner H. Dong X. Gabius H.J. Brewer C.F. Glycobiology. 1996; 6: 843-849Crossref PubMed Scopus (101) Google Scholar, 14Robbe C. Capon C. Coddeville B. Michalski J.C. Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (257) Google Scholar), whereas the x-ray crystal structures of CpCBM32 in complex with sugar help uncover the molecular details that confer this binding ability. Structural comparisons with known CBM32s and other C. perfringens CBM32s suggest variations in glycan specificities, but all are based on a key terminal galactose residue. This work provides the first detailed structure-function analysis of a family 32 CBM and will provide a foundation for further studies of CBMs within this family. Materials—Unless otherwise stated, chemicals, carbohydrates, glycoproteins, and polysaccharides were purchased from Sigma. Cloning—The DNA fragment encoding the family 32 CBM (Fig. 1) of CpGH84C was amplified by PCR from C. perfringens genomic DNA (Sigma, ATCC 13124) using previously described methods (15Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (20) Google Scholar). Nucleotides 1873-2301 of the cpgh84c gene, corresponding to the CBM (amino acid residues 625-767), were amplified with the oligonucleotide primers 5′-CACCAATCCAAGAACAGTAAAG-3′ (CBMF) and 5′-CTTTTATCCATGAACATTAACCTC-3′ (CBMR). The amplified gene fragment was ligated directly into the pET-150 TOPO Directional Cloning kit (Invitrogen) to generate pCBM32. The polypeptide (called CpCBM32) encoded by pCBM32 comprises a H6 tag fused to the CpCBM32 module by an enterokinase protease cleavage site. Protein Production and Purification—pCBM was transformed into the BL21star (DE3) Escherichia coli expression strain (Invitrogen). A 1.5-liter culture was grown in Luria-Bertani (LB) media, supplemented with ampicillin (100 μg/ml), to an optical density of ∼1 and induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside then grown overnight at 37 °C. The cells were harvested at 4,000 × g and resuspended in 20 ml of binding buffer containing 20 mm Tris, pH 8, and 0.5 m NaCl. Cells were lysed using a French pressure cell. Cell debris was removed by centrifugation for 1 h at 27,000 × g. The supernatant was applied to His-Select resin followed by step elution with binding buffer containing imidazole concentrations between 5 and 500 mm. Samples were run on a 15% SDS polyacrylamide gel, and fractions containing the polypeptide of interest were pooled. Proteins were concentrated, and buffer was exchanged in a stirred ultrafiltration unit (Amicon, Beverly, MA) using a 5,000 molecular weight cut-off membrane (Filtron, Northborough, MA). Purity, assessed by SDS-PAGE, was >95%. Determination of Protein Concentration—The concentrations of purified proteins were determined by UV absorbance (280 nm) using calculated molar extinction coefficients (16Mach H. Middaugh C.R. Lewis R.V. Anal. Biochem. 1992; 200: 74-80Crossref PubMed Scopus (429) Google Scholar). Macroarray Binding Experiments—CpCBM32 was labeled with Alexa Fluor 680 carboxylic acid, succinimidyl ester (Molecular Probes, Eugene, OR). Alexa Fluor 680 was resuspended in Me2SO to a concentration of 10 μg/μl. Negative and positive controls (bovine serum albumin and Clostridium cellulovorans CcCBM17, respectively) and CpCBM32 were buffer-exchanged into 0.2 m NaHCO3, pH 8.3, by de-salting using a PD-10 column (General Electric). 50 μg of Alexa Fluor 680 was incubated with 1 mg of protein, and the reaction was allowed to proceed for 1 h at room temperature in the dark. Buffer exchange was done into phosphate-buffered saline, pH 7.4, by gel-filtration using a PD-10 column to remove any unreacted dye molecules. 1 μl of 0.1 or 1% solutions of various plant polysaccharides, glycosaminoglycans, and glycoproteins were spotted onto a nitrocellulose membrane. Membranes were allowed to dry completely then blocked for 2 h with 10 ml of 1% bovine serum albumin, 0.05% Tween 20 in phosphate-buffered saline, pH 7.4. Labeled protein (0.4 mg) was allowed to incubate overnight at 13 °C in 10 ml of 1% Tween 20 in phosphate-buffered saline, pH 7.4. Blots were washed once with 10 ml of 1% Tween 20 in phosphate-buffered saline, pH 7.4, for 40 min. Emission of fluorescence was detected at 700 nm using the Odyssey Infrared Imaging System from LI-COR Biosciences. Detection was scored based on levels of signal emission (supplemental Fig. S1). UV Difference—Automated UV difference titrations of CpCBM32 were performed as described previously (17Boraston A.B. Warren R.A. Kilburn D.G. Biochemistry. 2001; 40: 14679-14685Crossref PubMed Scopus (32) Google Scholar). Difference spectra were examined for peak and trough wavelengths, and values at the appropriate wavelengths were extracted for further analysis. The wavelengths for the maximum peak-to-trough differences were determined individually for each sugar solution. The peak-to-trough heights at three wavelength pairs were calculated by subtraction of the trough values from the peak values, and the dilution-corrected data were plotted against total carbohydrate concentration. Data for the three wavelength pairs were analyzed simultaneously with MicroCal Origin (version 7.0) using a one-site binding model accounting for ligand depletion. Experiments were performed at 20 °C in 50 mm Tris, pH 7.5. The data reported are the averages and standard deviations of three independent titrations. Isothermal Titration Calorimetry—Isothermal titration calorimetry (ITC) was performed as described previously (18Boraston A.B. Creagh A.L. Alam M.M. Kormos J.M. Tomme P. Haynes C.A. Warren R.A. Kilburn D.G. Biochemistry. 2001; 40: 6240-6247Crossref PubMed Scopus (104) Google Scholar, 19Lammerts van Bueren A. Finn R. Ausio J. Boraston A.B. Biochemistry. 2004; 43: 15633-15642Crossref PubMed Scopus (36) Google Scholar) using a VP-ITC (MicroCal, Northampton, MA). Protein samples were extensively dialyzed against buffer (50 mm Tris, pH 7.5). Sugar solutions were prepared by mass in buffer saved from the final protein dialysis step. Both protein and sugar solutions were filtered and degassed immediately prior to use. Protein concentrations were determined by UV absorbance as described above. Aliquots of 10 mm LacNAc were titrated into CpCBM32 (350 μm), which gave C values >5 (20Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar). Aliquots of 5.0 mm type II H-trisaccharide were titrated into CpCBM32 (245 μm). In this case, the C value was <1(∼0.3) due to the low binding affinity. Based on the 1:1 binding observed in the crystal structure, the stoichiometry was fixed at 1 in the analysis of this data. Data were fit with a single binding site model. Crystallization and Data Collection—All crystallization experiments were performed using the hanging-drop vapor-diffusion method. Prior to crystallization, the H6 tag was removed from CpCBM32 by treatment with enterokinase over a 4-day period. The digested sample was run through a Novagen His-bind Quick 900 cartridge to remove the His tag and any undigested protein from the solution. Samples were concentrated and exchanged as above into 20 mm Tris, pH 8. Cocrystals of CpCBM32 (10.5 mg/ml) with galactose (∼10 mm) were obtained with 0.2 m MgCl2, 25% polyethylene glycol 2000 monomethylether, and 0.1 m Tris, pH 7.5. These crystals were cryoprotected with 15% glycerol in mother liquor. 1.5 m sodium/potassium phosphate was used to co-crystallize CpCBM32 (20 mg/ml) LacNAc and the type II blood group H-trisaccharide (α-l-fucosyl-1,2-β-d-galactosyl-1,4-β-d-N-acetylglucosamine). Optimization of this condition determined the ideal NaH2PO4/K2HPO4 concentration to be 1.5 m (at a 1:100 ratio) and the optimal protein concentration to be 5 mg/ml. The cryoprotectant used was 1.45 m Na/K2HPO4 with 27% ethylene glycol. Diffraction data were collected with a Rigaku R-AXIS IV++ area detector coupled to an MM-002 x-ray generator with Osmic “blue” optics and an Oxford Cryostream 700. Data were processed with Crystal Clear/d*trek (21Pflugrath J.W. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). All data collection statistics are given in Table 1.TABLE 1X-ray data collection and refinement statisticsDatasetGalactoseLacNAcH-trisaccharideSpace groupP3221P43212P43212Unit cella = 43.84, b = 43.84, c = 139.47; α = 90.00, β = 90.00, γ = 120.00a = 77.38, b = 77.38, c = 73.42; α = 90.00, β = 90.00, γ = 90.00a = 77.32, b = 77.32, c = 74.16; α = 90.00, β = 90.00, γ = 90.00,Asymmetric unit contentsMonomerMonomerMonomerResolution range20-1.49 (1.53-1.49)20-2.40 (2.49-2.40)20-2.30 (2.36-2.30)Number of measured reflections158,26939,83350,836Number of unique reflections23,6199,13010,441Rmerge (%)3.5 (33.7)9.9 (26.0)11.9 (30.7)Completeness (%)89.6 (50.1)99.6 (99.9)99.9 (99.5)I/σI21.6 (3.0)8.6 (4.0)7.8 (3.5)Redundancy6.70 (3.11)4.36 (4.42)4.87 (4.93)R value (%)20.617.518.1Rfree value (%)23.325.224.0r.m.s. bond lengths (Å)0.0180.0190.015r.m.s. bond angles (deg.)1.5441.6961.881r.m.s. chiral-centre restraints (Å3)0.1080.1070.088Overall B-factors (Å)All atoms22.4027.7930.67Protein atoms20.8826.5729.08Water molecules32.2434.7936.66Sugar16.3829.1345.64No. residues143142143No. waters172183218No. sugar atoms122636PDB code2j1a2j1e2j1f Open table in a new tab Structure Determination—Cp-CBM32 was solved by molecular replacement using the family 32 galactose-binding module from the M. viridifaciens sialidase (pdb code 1EUT) (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) as a search model. The program molrep (22Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) was able to find one clear rotation/translation solution corresponding to the single molecule in the asymmetric unit. This initial model was corrected, and ligand was added by successive rounds of building using COOT (23Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar). Refinement was done using REFMAC (24Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). Water molecules were added using the REFMAC implementation of ARP/wARP and inspected visually prior to deposition. The final model lacking waters was used as a starting model to solve the structures of the other CpCBM32 sugar complexes. Initial models were corrected, one or more ligands were added, and waters were added as above. Residue numbering conforms to the numbering in the complete CpGH84C enzyme. Final model statistics are given in Table 1. Carbohydrate Binding Properties of CpCBM32 from CpGH84C—Based on its amino acid sequence identity (∼30%) with the family 32 galactose-binding module from the M. viridifaciens sialidase we postulated that the CpCBM32 module in CpGH84C is indeed a carbohydrate-binding protein. This was initially investigated by macroarray binding experiments using arrayed glycoproteins and polysaccharides, which revealed significant binding to asialofetuin, type III porcine gastric mucin, and fetuin with relative binding of asialofetuin > porcine gastric mucin > fetuin (supplemental Fig. S1). Using knowledge of the glycan structures commonly found on these glycoproteins (14Robbe C. Capon C. Coddeville B. Michalski J.C. Biochem. J. 2004; 384: 307-316Crossref PubMed Scopus (257) Google Scholar) as a guide, we assessed the binding of various monosaccharides, disaccharides, and trisaccharides to CpCBM32 by qualitative and quantitative UV difference experiments. The addition of d-galactose and GalNAc (N-acetyl-d-galactosamine) to CpCBM32 resulted in large perturbations of the UV difference spectra indicative of the involvement of tryptophan in sugar binding (17Boraston A.B. Warren R.A. Kilburn D.G. Biochemistry. 2001; 40: 14679-14685Crossref PubMed Scopus (32) Google Scholar) (Fig. 2A). l-Fucose, d-glucose, d-mannose, and GlcNAc (N-acetyl-d-glucosamine) were also tested but did not influence the UV absorption of CpCBM32 and thus are unlikely primary ligands of CpCBM32. Quantitative studies by UV difference titrations showed an affinity of roughly 1 × 103 m-1 for galactose-based monosaccharides and a preference for β-rather than α-configured O-methylgalactose (Fig. 2B and Table 2). The presence of the acetamido group of GalNAc did not appear to confer any advantage to binding. CpCBM32 preferred lactose and LacNAc over galactose by factors of ∼2.5- and 10-fold, respectively. The increased affinity of LacNAc (cf. lactose) suggested the specific involvement of the 2′-acetamido group of the GlcNAc moiety in binding. Along similar lines, the α-1,2-linked l-fucose of fucosyllactose (α-l-fucosyl-1,2-β-d-galactosyl-1,4-β-d-glucose) substantially reduced the binding affinity to levels below those we could quantify due to limiting quantities of sugar but did not entirely legislate against binding.TABLE 2Binding constants determined by UV difference at 20 °C in 50 mm Tris-HCl, pH 7.5SugarKam–1 × 10–3d-Galactose0.98 (±0.17)d-GalNAc0.86 (±0.12)Methyl-α-d-galactose0.57 (±0.25)Methyl-β-d-galactose1.31 (±0.08)Lactose2.49 (±0.06)LacNAc9.09 (±2.98) Open table in a new tab The binding to LacNAc and the type II H-trisaccharide was further investigated by ITC (Fig. 2, C and D, and Table 3). The values for LacNAc revealed the enthalpically driven binding process common to protein-carbohydrate interactions (25Dam T.K. Brewer C.F. Chem. Rev. 2002; 102: 387-429Crossref PubMed Scopus (436) Google Scholar, 27Bachhawat-Sikder K. Thomas C.J. Surolia A. FEBS Lett. 2001; 500: 75-79Crossref PubMed Scopus (57) Google Scholar). The ΔH of binding was temperature-dependent, the analysis of which allowed the approximation of the change in heat capacity (ΔCp)to be -105 (±5) cal/mol/K. Again, this small negative ΔCp is consistent with the majority of protein-carbohydrate interactions. The affinity of CpCBM32 for the type II H-trisaccharide was too low to accurately deconvolute the stoichiometry of binding, so, on the basis of the LacNAc binding data and x-ray crystallography data (see below), this value was fixed at 1 for the analysis (28Turnbull W.B. Daranas A.H. J. Am. Chem. Soc. 2003; 125: 14859-14866Crossref PubMed Scopus (587) Google Scholar). Like with fucosyllactose, the fucose residue of the type II H-trisaccharide is detrimental to binding relative to LacNAc or lactose, although it does not destroy binding. The roughly 1.3 kcal/mol increase in free energy due to the fucose moiety on the H-trisaccharide appears to come by virtue of a substantial enthalpic penalty (∼+9.0 kcal/mol), which is partially offset by a favorable contribution to entropy (∼+25 cal/mol/K) (note: assumption of up to a 25% error in the estimate of stoichiometry due to errors in either the sugar or protein concentration does change the magnitudes of the calculated thermodynamic penalties but does not change their sign and, thus, qualitative interpretation of the data are unaffected).TABLE 3Binding parameters determined by ITC in 50 mm Tris-HCl, pH 7.5SugarTemperaturenKaΔHΔSΔG°C10–4 × m–1kcal/molcal/mol/Kkcal/molLacNAc250.89 ± 0.01.11 (±0.02)–15.6 (±0.0)–33.6 (±0.5)–5.5 (±0.0)20aOnly single titrations were performed, and errors are those from the data-fitting process. Otherwise, errors are the standard deviations determined from three independent experiments0.89 ± 0.01.77 (±0.02)–15.1 (±0.1)–32.1 (±0.5)–5.7 (±0.0)17.5aOnly single titrations were performed, and errors are those from the data-fitting process. Otherwise, errors are the standard deviations determined from three independent experiments0.89 ± 0.02.26 (±0.02)–14.8 (±0.1)–30.8 (±0.5)–5.8 (±0.0)H-trisaccharide25aOnly single titrations were performed, and errors are those from the data-fitting process. Otherwise, errors are the standard deviations determined from three independent experiments1.0bThis value was fixed as a constant for the data-fitting process0.12 (±0.00)–6.6 (±0.1)–8.3 (±0.1)–4.2 (±0.0)a Only single titrations were performed, and errors are those from the data-fitting process. Otherwise, errors are the standard deviations determined from three independent experimentsb This value was fixed as a constant for the data-fitting process Open table in a new tab The Fold of CpCBM32—CpCBM32 was crystallized in the presence of galactose, and its x-ray crystal structure was solved by molecular replacement at high resolution (1.49 Å). The fold is that of a β-sandwich comprising a β-sheet of three anti-parallel β-strands opposing a β-sheet of five anti-parallel β-strands (Fig. 3A). The closest structural neighbors are the family 32 CBMs from the fungal Cladobotryum dendroides galactose oxidase (PDB code 1GOF (9Ito N. Phillips S.E. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D. Knowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (697) Google Scholar)) (here the CBM is referred to as CdCBM32) and the bacterial M. viridifaciens sialidase (PDB code 1EUT (10Gaskell A. Crennell S. Taylor G. Structure. 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar)) (here the CBM is referred to as MvCBM32; see below for a more detailed comparison). More distantly related are the family 6 and 36 CBMs as well as the Anguilla sp. fucolectin. CpCBM32 heptahedrally coordinates one metal ion through the side chains of Thr-655, Asp-650, and Glu-672 and the backbone carbonyls of Phe-647, Asp-652, Thr-655, and Ala-761 (Fig. 3C). This ion was judged most likely to be calcium on the basis of three observations. Firstly, the atom possessed significant anomalous scattering properties, because it could be easily located in anomalous difference maps, indicating that the ion was not sodium or magnesium. Secondly, when considering ions commonly found associated with carbohydrate-binding proteins, heptahedral coordination mediated entirely by oxygen atoms is most consistent with potassium, calcium, or manganese. Lastly, when modeled as calcium, the B factor of this atom was consistent with the B factors of neighboring atoms in the coordinating side chains. This ion does not appear to play a direct role in binding carbohydrate, because it is quite distant from the carbohydrate binding site. Studies on other CBMs where similar “structurally” relevant ions have been removed have suggested only a stabilizing role for such ions, because apo-CBMs are still competent to bind carbohydrate (29Roske Y. Sunna A. Pfeil W. Heinemann U. J. Mol. Biol. 2004; 340: 543-554Crossref PubMed Scopus (16) Google Scholar, 30Johnson P.E. Creagh A.L. Brun E. Joe K. Tomme P. Haynes C.A. McIntosh L.P. Biochemistry. 1998; 37: 12772-12781Crossref PubMed Scopus (21) Google Scholar). It is notable that the structural position of this ion is also conserved in CBMs from family 6 (7Boraston A.B. Notenboom V. Warren R.A. Kilburn D.G. Rose D.R. Davies G. J. Mol. Biol. 2003; 327: 659-669Crossref PubMed Scopus (64) Google Scholar, 31Morland C. van Bueren A.L. Gilbert H.J. Boraston A.B. J. Biol. Chem. 2005; 280: 530-537Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 32Lammerts van Bueren A. Boraston A.B. J. Mol. Biol. 2004; 340: 869-879Crossref PubMed Scopus (20) Google Scholar), 36 (33Jamal-Talabani S. Boraston A.B. Turkenburg J.P. Tarbouriech N. Ducros V.M. Davies G.J. Structure. 2004; 12: 1177-1187Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and the Anguilla sp. fucolectins (34Bianchet M.A. Odom E.W. Vasta G.R. Amzel L.M. Nat. Struct. Biol. 2002; 9: 628-634PubMed Google Scholar). CpCBM32 in Complex with Carbohydrates—The electron density map of the galactose complex clearly revealed a single bound molecule of galactose. Trp-661 and Phe-757 create a relatively hydrophobic pocket, which cradles the C6-hydroxymethyl group. Trp-661 “stacks” against the flat, apolar surface created by carbons 3-6 on the B-face of d-galactose with the O4 pointing away from the aromatic residue (Fig. 4). Specificity for the non-reducing end of d-galactose (and presumably GalNAc) is conferred by three potential hydrogen bonds to the axial O4 of the sugar from the terminal δO of Asn-695, a terminal guanido nitrogen of Arg-690, and the ϵN from the imidazole ring of His-658 (Fig. 5). Additional hydrogen bonds are made between the O3 of d-galactose and Arg-690 and Glu-641; the endocyclic oxygen of galactose makes a h" @default.
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• W1983125435 title "The Interaction of a Carbohydrate-binding Module from a Clostridium perfringens N-Acetyl-β-hexosaminidase with Its Carbohydrate Receptor" @default.
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