The Family 6 Carbohydrate Binding Module CmCBM6-2 Contains Two Ligand-binding Sites with Distinct Specificities
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m401620200
ISSN1083-351X
AutoresJ. Henshaw, David N. Bolam, Virgínia M. R. Pires, Mirjam Czjzek, Bernard Henrissat, L.M.A. Ferreira, C.M.G.A. Fontes, Harry J. Gilbert,
Tópico(s)Biofuel production and bioconversion
ResumoThe microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the β1,4-β1,3-mixed linked glucans lichenan and barley β-glucan, cello-oligosaccharides, insoluble forms of cellulose, the β1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the β1,4,-β1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site. The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the β1,4-β1,3-mixed linked glucans lichenan and barley β-glucan, cello-oligosaccharides, insoluble forms of cellulose, the β1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the β1,4,-β1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site. Microbial degradation of the plant cell wall is the primary mechanism by which organic carbon is recycled in the biosphere. The plant cell wall is a complex insoluble structure that is highly recalcitrant to biological attack (1Brett C.T. Waldren K. Black M. Charlewood B. Physiology and Biochemistry of Plant Cell Walls: Topics in Plant Functional Biology. 1. Chapman & Hall, London1996Google Scholar, 2Tomme P. Warren R.A. Gilkes N.R. Adv. Microb. Physiol. 1995; 37: 1-81Crossref PubMed Scopus (625) Google Scholar). To increase the efficiency of this degradative process microorganisms synthesize modular plant cell wall hydrolases in which the catalytic modules are attached via linker peptides to non-catalytic carbohydrate binding modules (CBMs 1The abbreviations used are: CBM, carbohydrate-binding module; CsCBM, CBM from a Clostridium stercorarium putative xylanase; CtCBM, CBM from Clostridium thermocellum Xyn11A; d.p., degree of polymerization; G2, cellobiose; G3, cellotriose; G6, cellohexaose; Cel5A, endoglucanase 5A; HEC, hydroxyethyl cellulose. (3Boraston A.B. McLean B.W. Kormos J. Alam M.M. Gilkes N.R. Haynes C.A. Tomme P. Kilburn D.G. Warren R.A. H.J. G. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge, UK1999: 202-211Google Scholar)). CBMs potentiate catalytic activity by mediating prolonged and intimate association between the enzyme and its target substrate (4Bolam 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 (237) Google Scholar, 5Gill 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), whereas some of these modules also increase substrate access by disrupting the crystalline structure of cellulose (6Din N. Damude H.G. Gilkes N.R. Miller Jr., R.C. Warren R.A. Kilburn D.G. Proc. Natl. Acad. Sci. U. S. 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It should be noted, however, that many plant cell wall hydrolases that do not hydrolyze cellulose contain CBMs that interact specifically with the crystalline form of this polysaccharide (2Tomme P. Warren R.A. Gilkes N.R. Adv. Microb. Physiol. 1995; 37: 1-81Crossref PubMed Scopus (625) Google Scholar, 10Boraston A.B. Revett T.J. Boraston C.M. Nurizzo D. Davies G.J. Structure. 2003; 11: 665-675Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 11Hogg D. Pell G. Dupree P. Goubet F. Martin-Orue S.M. Armand S. Gilbert H.J. Biochem. J. 2003; 371: 1027-1043Crossref PubMed Google Scholar, 12Ferreira L.M. Durrant A.J. Hall J. Hazlewood G.P. Gilbert H.J. Biochem. J. 1990; 269: 261-264Crossref PubMed Scopus (74) Google Scholar). Based on primary structural similarities CBMs have been grouped into 34 families (afmb.cnrs-mrs.fr/CAZY (13Coutinho P.M. Henrissat B. Gilbert J.J. Davies G. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge, UK1999: 3-12Google Scholar)). Although the ligand specificity of families 1, 5, and 10, which bind to crystalline polysaccharides, is invariant, there are considerable differences in carbohydrate recognition in other CBM families. For example, the CBM2 family contains proteins that bind to xylan (14Black G.W. Hazlewood G.P. Millward-Sadler S.J. Laurie J.I. Gilbert H.J. Biochem. J. 1995; 307: 191-195Crossref PubMed Scopus (90) Google Scholar) and crystalline cellulose (12Ferreira L.M. Durrant A.J. Hall J. Hazlewood G.P. Gilbert H.J. Biochem. J. 1990; 269: 261-264Crossref PubMed Scopus (74) Google Scholar), whereas different members of CBM4 recognize laminarin, individual cellulose chains, and xylan (8Abou 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, 9Boraston 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 (128) Google Scholar), (15Zverlov V.V. Volkov I.Y. Velikodvorskaya G.A. Schwarz W.H. Microbiology. 2001; 147: 621-629Crossref PubMed Scopus (46) Google Scholar). The CBMs described to date are β-stranded proteins in which the topology of the carbohydrate-binding site reflects the nature of the target ligand. For example, CBMs that bind to crystalline cellulose (known as type A modules) contain a planar hydrophobic ligand binding surface (16Kraulis J. Clore G.M. Nilges M. Jones T.A. Pettersson G. Knowles J. Gronenborn A.M. Biochemistry. 1989; 28: 7241-7257Crossref PubMed Scopus (489) Google Scholar, 17Raghothama S. Simpson P.J. Szabo L. Nagy T. Gilbert H.J. Williamson M.P. Biochemistry. 2000; 39: 978-984Crossref PubMed Scopus (68) Google Scholar, 18Xu G.Y. Ong E. Gilkes N.R. Kilburn D.G. Muhandiram D.R. Harris-Brandts M. Carver J.P. Kay L.E. Harvey T.S. Biochemistry. 1995; 34: 6993-7009Crossref PubMed Scopus (214) Google Scholar, 19Tormo J. Lamed R. Chirino A.J. Morag E. Bayer E.A. Shoham Y. Steitz T.A. EMBO J. 1996; 15: 5739-5751Crossref PubMed Scopus (414) Google Scholar). By contrast, CBMs that interact with individual polysaccharide chains (type B modules) accommodate their target ligands in a cleft of varying depth (9Boraston 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 (128) Google Scholar, 20Charnock S.J. Bolam D.N. Turkenburg J.P. Gilbert H.J. Ferreira L.M. Davies G.J. Fontes C.M. Biochemistry. 2000; 39: 5013-5021Crossref PubMed Scopus (151) Google Scholar, 21Czjzek 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 (100) Google Scholar). In general both type A and type B CBMs interact with five or six saccharide units via hydrophobic stacking interactions between alternate sugar rings and aromatic amino acids, although hydrogen bonds also play an important role in carbohydrate recognition by type B modules (22Xie 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, 23Pell G. Williamson M.P. Walters C. Du H. Gilbert H.J. Bolam D.N. Biochemistry. 2003; 42: 9316-9323Crossref PubMed Scopus (55) Google Scholar). The three-dimensional structure of almost all type B CBMs determined to date conform to a classic lectin-like β-jelly roll in which a single ligand-binding site comprises a shallow cleft on the concave surface of the protein. In CBM4 and CBM2, variation in ligand recognition between different members of these families is reflected in the structure of this conserved binding site (9Boraston 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 (128) Google Scholar). Although two xylan binding CBM6s from Clostridium thermocellum Xyn11A (CtCBM6) and a Clostridium stercorarium putative xylanase (CsCBM6-3) also display a β-jelly roll fold with a potential ligand-binding site (cleft B) on the concave surface, a second possible binding site (cleft A) is located on one edge of the protein between the loops that connect the inner and outer β-sheets (21Czjzek 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 (100) Google Scholar). Mutagenesis, structural, and NMR studies revealed that in CtCBM6 and CsCBM6-3 only cleft A binds ligand, whereas cleft B is unable to interact with carbohydrates, as it appears to be partially occluded by a proline-containing loop in both CBMs (21Czjzek 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 (100) Google Scholar, 24Boraston 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). It was proposed that the variation in the location of the functional binding site(s) in the different members of this family may contribute to the diversity of ligand specificity observed within CBM6, which contains proteins that bind to cellulose and xylan (21Czjzek 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 (100) Google Scholar). To test this hypothesis a biochemical (this study) and structural approach (accompanying paper, Ref. 25Pires V.M.R. Henshaw J. Prates J.A.M. Bolam D. Ferreira L.M.A. Fontes C.M.G.A. Henrissat B. Planas A. Gilbert H.J. Czjzek M. J. Biol. Chem. 2004; 279: 21560-21568Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) was employed to dissect the ligand specificity of the C-terminal CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A (Cel5A; Ref. 26Fontes C.M. Clarke J.H. Hazlewood G.P. Fernandes T.H. Gilbert H.J. Ferreira L.M. Appl. Microbiol. Biotechnol. 1998; 49: 552-559Crossref PubMed Scopus (16) Google Scholar). The data show that the protein can accommodate β1,4- and β1,3-linked glucans in cleft A and B and that the two binding sites mediate binding to insoluble cellulose. Only cleft B, however, interacts with β1,3-β1,4-mixed linked glucans such as lichenan and barley β-glucan, supporting the hypothesis that the two potential binding clefts contribute to the variation in ligand specificity observed within the CBM6 family. Source of Carbohydrates Used—All oligosaccharides and polysaccharides were purchased from Megazyme International (Bray County Wicklow, Ireland), except oat spelt xylan and hydroxyethylcellulose, which were obtained from Sigma, and cello-oligosaccharides, which were from Seikagaku Corp. (Japan). Acid-swollen cellulose was prepared from Avicel (PH101; Serva) as described previously (27Wood T.M. Methods Enzymol. 1988; 160: 19-25Crossref Scopus (247) Google Scholar). Protein Expression and Purification—The regions of the CmCel5A gene (cel5A; Ref. 26Fontes C.M. Clarke J.H. Hazlewood G.P. Fernandes T.H. Gilbert H.J. Ferreira L.M. Appl. Microbiol. Biotechnol. 1998; 49: 552-559Crossref PubMed Scopus (16) Google Scholar)) encoding CmCBM6-2 were amplified by the PCR using the primers 5′-CTCCATATGGTAATCGCGACTATTC-3′ and 5′-CACGGATCCTTAATGTGTCTTGTTG-3′ (amplifies nucleotides 1475-1869 of cel5A). The reverse primer contains the cel5A stop codon. The amplified DNA was digested with NdeI and XhoI (restriction sites, shown in bold, were incorporated into the two primers) and cloned into the similarly restricted expression vector pET28a to generate pCF1. CmCBM6-2, encoded by pCF1, contains an N-terminal His6 tag. To express CmCBM6-2 the Escherichia coli strain Tuner (Novagen), harboring pCF1, was cultured in Luria Bertani broth containing 10 μg ml-1 kanamycin at 37 °C to mid-exponential phase (A600 ∼0.3). The temperature was then reduced to 16 °C, and when the absorbance had reached 0.7 isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 200 μm, and the cultures were incubated for a further 16 h. Cell-free extracts were then prepared, and recombinant Cm- CBM6-2 was purified by immobilized metal ion affinity chromatography using TALON resin (Clontech) as described previously (22Xie 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), except that 5 mm CaCl2 was included in all buffers. Site-directed Mutagenesis—Mutants of CmCBM6-2 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The primers used to generate these mutants are listed in Table I, and each of the mutated CBM6 genes was sequenced by MWG Biotech (Germany) to ensure that only the appropriate mutations had been incorporated.Table IMutagenic primers used in this studyMutantPrimerQ13A, F5′-GAAGACCATAGCGCACAGAGCGGCAC-3′Q13A, R5′-GTGCCGCTCTGTGCGCTATGGTCTTC-3′Y33A, F5′-CGGTGGTAAAAACGTTGGCGCTATCGATGCCGGGGAC-3′Y33A, R5′-GTCCCCGGCATCGATAGCGCCAACGTTTTTACCACCG-3′S41A, F5′-GGGGACTGGCTCGCTTACGCGGGTACG-3′S41A, R5′-CGTACCCGCGTAAGCGAGCCAGTCCC-3′R60A, F5′-GCTATTTGATTGAGTACGCTGTGGCCAGCC-3′R60A, R5′-GGCTGGCCACAGCGTACTCAATCAAATAGC-3′E73A, F5′-CAGCCTGACATTTGCAGAAGCAGGCGG-3′E73A, R5′-CCGCCTGCTTCTGCAAATGTCAGGCTG-3′W92A, F5′-GCAACCGGTGGCGCGCAAACCTGGACG-3′W92A, R5′-CGTCCAGGTTTGCGCGCCACCGGTTGC-3′K114A, F5′-CCATCAATTTGGGATTGCAGCCAATGCCGG-3′K114A, R5′-CCGGCATTGGCTGCAATCCCAAATTGATGG-3′N116A, F5′-GGGATTAAAGCCGCTGCCGGTGGTTG-3′N116A, R5′-CAACCACCGGCAGCGGCTTTAATCCC 3′ Open table in a new tab Affinity Gel Electrophoresis—Qualitative assessment of the capacity of CmCBM6-2 to bind to soluble polysaccharides was determined by affinity gel electrophoresis. The method was essentially as described by Freelove et al. (28Freelove 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 the polysaccharide ligands at a concentration of 0.1% (w/v), and electrophoresis was carried out for 4 h. The non-binding protein, which was used as a negative control, was bovine serum albumin. When CmCBM6-2 bound to a polysaccharide ligand it could be visualized as a tight, but very slow migrating band, whereas in the gels lacking ligand (or ligand that the protein did not interact with) the CBM ran as a smear making it difficult to visualize. Isothermal Titration Calorimetry (ITC)—ITC measurements were made following standard procedures (20Charnock S.J. Bolam D.N. Turkenburg J.P. Gilbert H.J. Ferreira L.M. Davies G.J. Fontes C.M. Biochemistry. 2000; 39: 5013-5021Crossref PubMed Scopus (151) Google Scholar) using a Microcal Omega titration calorimeter. The proteins were dialyzed extensively against 50 mm Na-HEPES buffer, pH 7.0, 5 mm CaCl2, and the ligand was dissolved in the same buffer to minimize heats of dilution. During a titration experiment the protein sample stirred at 300 rpm in a 1.4331-ml reaction cell was injected with 25-50 successive 10-μl aliquots of polysaccharide (5-20 mg ml-1) or oligosaccharide (7.5-20 mm) ligand at 200-s intervals. The molar concentration of CmCBM6-2-binding sites present in the polysaccharide ligands was determined as described previously (31Szabo 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 (85) 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), number of binding sites on the protein (n), and the enthalpy of binding (ΔH). Other thermodynamic parameters were calculated using the standard thermodynamic equation-RTln KA = ΔG = ΔH-TΔS. Titrations were carried out in triplicate for most ligands, and the errors are the S.D. of the mean of these replicates. Binding to Insoluble Polysaccharides—Qualitative assessment of binding to insoluble cellulose was carried out as follows: 200 μl of a 250 μg ml-1 solution of CmCBM6-2 in 20 mm Tris-HCl, pH 8.0, containing 300 mm NaCl and 5% (v/v) Tween 20 (Buffer A) was mixed with 2 mg of insoluble polysaccharide. The reaction mixture was incubated on ice for 1 h with occasional mixing, after which the insoluble cellulose was pelleted by centrifugation at 13,000 × g for 2 min. The supernatant, comprising the unbound fraction, was removed, and all samples were analyzed by SDS-PAGE using a 12.5% gel. Controls with protein but no cellulose were included to ensure that no precipitation occurred during the assay period. Depletion isotherms to quantify the binding of wild type and mutants of CmCBM6-2 to both acid-swollen cellulose and Avicel were carried out as follows: protein (250 μl in Buffer A) at concentrations between 1 and 100 μm was added to 0.25 mg of cellulose and incubated on ice for 1 h with gentle mixing. The polysaccharide was then centrifuged at 13,000 × g for 1 min, and the A280 of the supernatant was measured to quantify the amount of free protein remaining after binding. Bound protein was calculated from the total minus the free. Controls with protein but no cellulose were included to ensure that no precipitation occurred during the assay period. The data were analyzed by nonlinear regression using a standard one-site binding model (GraphPad Prism, v2.01), and the Bmax (amount of CBM bound at saturation) and KA values were obtained from the regressed isotherm data. At least three separate binding isotherms were carried out for each protein. Ligand Specificity of CmCBM6-2—Cel5A contains an N-terminal GH5 catalytic domain and two family 6 CBMs, with the C-terminal module designated CmCBM6-2 (26Fontes C.M. Clarke J.H. Hazlewood G.P. Fernandes T.H. Gilbert H.J. Ferreira L.M. Appl. Microbiol. Biotechnol. 1998; 49: 552-559Crossref PubMed Scopus (16) Google Scholar). To evaluate the ligand binding specificity of CmCBM6-2, the protein module was expressed in E. coli and purified to electrophoretic homogeneity (Fig. 1). Affinity gel electrophoresis, Table II, shows that CmCBM6-2 binds to the β1,4-β1,3-mixed-linked glucans lichenan and barley β-glucan and the β1,3-glucan laminarin but does not display significant affinity for poorly substituted or highly decorated xylans, pectins such as polygalacturonic acid or rhamnogalacturonan I, potato or pectic β1,4-galactan, sugar beet arabinan, locust bean or carob galactomannan, and konjac glucomannan.Table IILigand specificity of wild type and mutants of CmCBM6-2, determined by affinity gel electrophoresisProteinPolysaccharideBindingaBinding was evaluated by affinity gel electrophoresis as described under "Experimental Procedures." Polysaccharide was added to the ligand gel at a final concentration of 0.1% (w/v), and bovine serum albumin was used as the non-binding negative control.Wild typeBarley β-glucan+b+, binding; −, no binding.Wild typeLichenan+Wild typeLaminarin+Wild typeHydroxyethylcellulose−b+, binding; −, no binding.Wild typeKonjac glucomannan−Wild typeCarob galactomannan−Wild typeOat spelt xylan−Wild typeWheat arabinoxylan−Wild typeLinear α1,5-arabinan−Wild typePotato β1,4-galactan−Wild typeXyloglucan−Wild typeRhamnogalacturonan I−Wild typePolygalacturonic acid−Y33AcMutation in cleft A.Barley β-glucan+Y33AcMutation in cleft A.Lichenan+Y33AcMutation in cleft A.Laminarin+W92AcMutation in cleft A.Barley β-glucan+W92AcMutation in cleft A.Lichenan+W92AcMutation in cleft A.Laminarin+33A/W92AcMutation in cleft A.Barley β-glucan+W92A/Y33AcMutation in cleft A.Lichenan+W92A/Y33AcMutation in cleft A.Laminarin+W39AdMutation in cleft B.Barley β-glucan−W39AdMutation in cleft B.Lichenan−W39AdMutation in cleft B.Laminarin+E73AdMutation in cleft B.Barley β-glucan−E73AdMutation in cleft B.Lichenan−E73AdMutation in cleft B.Laminarin+Q13AdMutation in cleft B.Barley β-glucan+Q13AdMutation in cleft B.Lichenan+Q13AdMutation in cleft B.Laminarin+K114AdMutation in cleft B.Barley β-glucan+K114AdMutation in cleft B.Lichenan+K114AdMutation in cleft B.Laminarin+a Binding was evaluated by affinity gel electrophoresis as described under "Experimental Procedures." Polysaccharide was added to the ligand gel at a final concentration of 0.1% (w/v), and bovine serum albumin was used as the non-binding negative control.b +, binding; −, no binding.c Mutation in cleft A.d Mutation in cleft B. Open table in a new tab ITC was used to quantify the interaction of CmCBM6-2 with its target ligands. Representative titrations that can be deconvoluted to give thermodynamic and binding parameters are displayed in Fig. 2, and the full data set is presented in Table III. ITC data that could only provide qualitative information on the binding of some ligands because the affinities were too low to obtain sigmoidal titration curves, are displayed in Table IV, and examples of these titrations are provided in the Supplemental Fig. 1. CmCBM6-2 binds to β1,3-β1,4 and mixed-linked β1,4-β1,3-glucans, and to xylooligosaccharides (Table III and Fig. 2) with affinities that are similar to most Type B CBMs from mesophilic microorganisms (29Xie H. Bolam D.N. Nagy T. Szabo L. Cooper A. Simpson P.J. Lakey J.H. Williamson M.P. Gilbert H.J. Biochemistry. 2001; 40: 5700-5707Crossref PubMed Scopus (57) Google Scholar, 30Boraston A.B. Tomme P. Amandoron E.A. Kilburn D.G. Biochem. J. 2000; 350: 933-941Crossref PubMed Google Scholar, 31Szabo 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 (85) Google Scholar, 32Charnock 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 (84) Google Scholar). The stoichiometry for the binding of CmCBM6-2 to cellohexaose is close to 1:2, indicating that the protein module contains two distinct ligand-binding sites that can each accommodate this oligosaccharide. The isotherms, however, display only one obvious binding event, and the data fit a single-site model well, suggesting that the two sites are non-interacting and have similar affinities for cellohexaose. It was not possible, therefore, to deconvolute the ITC data (for the wild type protein) to determine the individual affinity for the oligosaccharide ligand at each discrete binding site. This issue was resolved using a mutagenesis approach described below.Table IIIAffinity of CmCBM6-2 and its derivatives for oligosaccharides and polysaccharides determined by ITCProteinLigandKA × 10−3ΔGΔHTΔSnan is the number of binding sites on the protein.m−1kcal mol−1kcal mol−1kcal mol−1Wild typeβ-Glucan8.6 ± 1.4−5.0 ± 0.1−9.0 ± 0.5−4.0 ± 0.71.0 ± 0.0Wild typeLichenan7.8 ± 0.5−4.9 ± 0.0−11.2 ± 0.3−6.3 ± 0.41.0 ± 0.1Wild typeCellohexaose6.8 ± 0.4−4.9 ± 0.0−9.8 ± 0.2−4.9 ± 0.21.6 ± 0.1Wild typeLaminarin8.0 ± 0.8−5.0 ± 0.0−15.9 ± 0.5−10.9 ± 0.51.8 ± 0.4Wild typeLaminohexaose5.1 ± 0.5−4.7 ± 0.0−15.1 ± 0.3−10.4 ± 0.32.4 ± 0.3W92A/Y33Aβ-Glucan6.8 ± 0.3−4.9 ± 0.0−9.2 ± 0.2−4.3 ± 0.11.1 ± 0.0W92A/Y33ALichenan8.4 ± 1.3−5.0 ± 0.1−11.1 ± 0.2−6.1 ± 0.31.1 ± 0.1W92A/Y33ACellohexaose12.1 ± 0.2−5.2 ± 0.0−8.3 ± 0.0−3.1 ± 0.10.9 ± 0.0W92A/Y33ACellopentaose10.8 ± 0.4−5.1 ± 0.0−8.1 ± 0.1−3.0 ± 0.21.1 ± 0.1W92A/Y33ACellotetraose11.1 ± 0.3−5.1 ± 0.0−8.4 ± 0.2−3.3 ± 0.20.8 ± 0.0W92A/Y33ACellotriose12.7 ± 0.2−5.2 ± 0.0−8.1 ± 0.1−2.9 ± 0.10.9 ± 0.0E73ACellohexaose5.9 ± 0.4−4.8 ± 0.0−9.2 ± 0.4−4.4 ± 0.50.7 ± 0.0E73ACellopentaose7.1 ± 0.4−4.9 ± 0.0−9.3 ± 0.2−4.4 ± 0.30.6 ± 0.2E73ACellotetraose7.0 ± 0.2−4.9 ± 0.0−9.1 ± 0.2−4.2 ± 0.20.6 ± 0.1E73ACellotriose5.9 ± 0.5−4.8 ± 0.0−9.2 ± 0.1−4.4 ± 0.20.6 ± 0.1E73ACellobiose8.1 ± 0.3−5.0 ± 0.0−9.1 ± 0.1−4.1 ± 0.10.6 ± 0.1E73AXylohexaose4.6 ± 0.7−4.7 ± 0.1−7.5 ± 0.4−2.8 ± 0.51.0 ± 0.1E73AXylobiose5.1 ± 0.4−4.7 ± 0.0−7.2 ± 0.3−2.5 ± 0.41.2 ± 0.0E73ALaminarin5.0 ± 1.1−4.7 ± 0.2−10.5 ± 0.6−5.8 ± 0.80.9 ± 0.1E73ALaminohexaose4.8 ± 0.3−4.7 ± 0.1−8.7 ± 0.4−4.0 ± 0.60.8 ± 0.2Q13Aβ-Glucan7.1 ± 0.5−4.9 ± 0.0−8.8 ± 0.2−3.9 ± 0.31.3 ± 0.3Q13ALichenan5.2 ± 0.4−4.7 ± 0.0−11.0 ± 0.5−6.3 ± 0.61.0 ± 0.2Q13ACellohexaose6.0 ± 0.5−4.8 ± 0.0−10.2 ± 0.2−5.4 ± 0.21.8 ± 0.0Q13ALaminarin8.4 ± 0.9−5.0 ± 0.1−16.0 ± 0.3−11.0 ± 0.51.2 ± 0.1K114Aβ-Glucan8.3 ± 0.3−5.0 ± 0.0−7.8 ± 0.1−2.8 ± 0.20.9 ± 0.3K114ALichenan8.7 ± 0.6−5.0 ± 0.0−11.3 ± 0.4−6.3 ± 0.50.8 ± 0.2K114ACellohexaose8.2 ± 0.4−5.0 ± 0.0−9.6 ± 0.2−4.6 ± 0.22.1 ± 0.1a n is the number of binding sites on the protein. Open table in a new tab Table IVQualitative assessment of binding of CmCBM6-2 mutants to gluco-and xylo-configured saccharides as determined by ITCProteinLigandBindingW92A/Y33ALaminohexaose+aA plus symbol (+) indicates binding. Although the affinity was too low to accu
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