Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study.

The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods. CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose. The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids. Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft. In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs. The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces.

beta-1,3-Glucan binding by a thermostable carbohydrate-binding module from Thermotoga maritima.

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T. maritima comprise a highly thermostable family 4 CBM that binds beta-1,3- and beta-(1,3)(1,4)-glucans. Laminarin, a beta-1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 x 10(7) M(-1)) and one with a 100-fold lower affinity (2.4 x 10(5) M(-1)). The affinities for laminarioligosaccharides and beta-(1,3)(1,4)-glucans ranged from approximately 2 x 10(5) to approximately 2.5 x 10(6) M(-1). Cellooligosaccharides and laminariobiose were bound only very weakly (K(a)s approximately 5 x 10(3) M(-1)). Spectroscopic and mutagenic studies implicated the involvement of three tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with large negative changes in heat capacity. Temperature and osmotic conditions profoundly influenced binding. For the first time in solution, the direct uptake and release of water in CBM binding are demonstrated.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis.

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A.

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.

Crystal structures of the family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A in native and ligand-bound forms.

The C-terminal module of the thermostable Thermotoga maritima xylanase 10A (CBM9-2) is a family 9 carbohydrate-binding module that binds to amorphous and crystalline cellulose and a range of soluble di- and monosaccharides as well as to cello and xylo oligomers of different degrees of polymerization [Boraston, A. B., Creagh, A. L., Alam, Md. M., Kormos, J. M., Tomme, P., Haynes, C. A., Warren, R. A. J., and Kilburn, D. G. (2001) Biochemistry 40, 6240-6247]. The crystal structure of CBM9-2 has been determined by the multiwavelength anomalous dispersion method to 1.9 A resolution. CBM9-2 assumes a beta-sandwich fold and contains three metal binding sites. The bound metal atoms, which are most likely calcium cations, are in an octahedral coordination. The crystal structures of CBM9-2 in complex with glucose and cellobiose were also determined in order to identify the sugar-binding site and provide insight into the structural basis for sugar binding by CBM9-2. The sugar-binding site is a solvent-exposed slot sufficient in depth, width, and length to accommodate a disaccharide. Two tryptophan residues are stacked together on the surface of the protein forming the sugar-binding site. From the complex structures with glucose and cellobiose, it was inferred that CBM9-2 binds exclusively to the reducing end of mono-, di-, and oligosaccharides with an intricate hydrogen-bonding network involving mainly charged residues, as well as stacking interactions by Trp175 and Trp71. The binding interactions are limited to disaccharides as was expected from calorimetric data. Comparison of the glucose and cellobiose complexes revealed surprising differences in binding of these two substrates by CBM9-2. Cellobiose was found to bind in a distinct orientation from glucose, while still maintaining optimal stacking and electrostatic interactions with the reducing end sugar.

A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris.

The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.

Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A.

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1). Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.

A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A.

The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of approximately 1x10(2) M(-1)-1x10(3) M(-1). It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2+/-0. 6)x10(3)/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the alpha, beta and gamma sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only approximately (0. 5+/-0.1)x10(3)/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.

Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.