Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.     

Family 6 carbohydrate binding modules recognize the non-reducing end of beta-1,3-linked glucans by presenting a unique ligand binding surface

Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the beta-1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display beta-1,3-glucanase activity. BhCBM6 binds beta-1,3-glucooligosaccharides with affinities of approximately 1 x 10(5) m(-1). The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended beta-1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site.     

Carbohydrate-binding domains: multiplicity of biological roles

Insoluble polysaccharides can be degraded by a set of hydrolytic enzymes formed by catalytic modules appended to one or more non-catalytic carbohydrate-binding modules (CBM). The most recognized function of these auxiliary domains is to bind polysaccharides, bringing the biocatalyst into close and prolonged vicinity with its substrate, allowing carbohydrate hydrolysis. Examples of insoluble polysaccharides recognized by these enzymes include cellulose, chitin, β-glucans, starch, glycogen, inulin, pullulan, and xylan. Based on their amino acid similarity, CBMs are grouped into 55 families that show notable variation in substrate specificity; as a result, their biological functions are miscellaneous. Carbohydrate or polysaccharide recognition by CBMs is an important event for processes related to metabolism, pathogen defense, polysaccharide biosynthesis, virulence, plant development, etc. Understanding of the CBMs properties and mechanisms in ligand binding is of vital significance for the development of new carbohydrate-recognition technologies and provide the basis for fine manipulation of the carbohydrate–CBM interactions.     

Family 6 carbohydrate binding modules in beta-agarases display exquisite selectivity for the non-reducing termini of agarose chains

Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.     

Characterization of surface binding sites in glycoside hydrolases: A computational study

Structural properties of carbohydrate surface binding sites (SBSs) were investigated with computational methods. Eighty‐five SBSs of 44 enzymes in 119 Protein Data Bank (PDB) files were collected as a dataset. On the basis of SBSs shape, they were divided into 3 categories: flat surfaces, clefts, and cavities (types A, B, and C, respectively). Ligand varieties showed the correlation between shape of SBSs and ligands size. To reduce cut‐off differences in each SBSs with different ligand size, molecular docking were performed. Molecular docking results were used to refine SBSs classification and binding sites cut‐off. Docking results predicted putative ligands positions and displayed dependence of the ligands binding mode to the structural type of SBSs. Physicochemical properties of SBSs were calculated for all docking results with YASARA Structure. The results showed that all SBSs are hydrophilic, while their charges could vary and depended to ligand size and defined cut‐off. Surface binding sites type B had highest average values of solvent accessible surface area. Analysis of interactions showed that hydrophobic interactions occur more than hydrogen bonds, which is related to the presence of aromatic residues and carbohydrates interactions.     

Non-catalytic Binding Sites Induce Weaker Long-Range Evolutionary Rate Gradients than Catalytic Sites in Enzymes

Enzymes exhibit a strong long-range evolutionary constraint that extends from their catalytic site and affects even distant sites, where site-specific evolutionary rate increases monotonically with distance. While protein–protein sites in enzymes were previously shown to induce only a weak conservation gradient, a comprehensive relationship between different types of functional sites in proteins and the magnitude of evolutionary rate gradients they induce has yet to be established. Here, we systematically calculate the evolutionary rate (dN/dS) of sites as a function of distance from different types of binding sites in enzymes and other proteins: catalytic sites, non-catalytic ligand binding sites, allosteric binding sites, and protein–protein interaction sites. We show that catalytic sites indeed induce significantly stronger evolutionary rate gradient than all other types of non-catalytic binding sites. In addition, catalytic sites in enzymes with no known allosteric function still induce strong long-range conservation gradients. Notably, the weak long-range conservation gradients induced by non-catalytic binding sites in enzymes is nearly identical in magnitude to those induced by ligand binding sites in non-enzymes. Finally, we show that structural determinants such as local solvent exposure of sites cannot explain the observed difference between catalytic and non-catalytic functional sites. Our results suggest that enzymes and non-enzymes share similar evolutionary constraints only when examined from the perspective of non-catalytic functional sites. Hence, the unique evolutionary rate gradient from catalytic sites in enzymes is likely driven by the optimization of catalysis rather than ligand binding and allosteric functions.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

Analysis of the surfaces of wood tissues and pulp fibers using carbohydrate-binding modules specific for crystalline cellulose and mannan

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1-->4)-beta-mannan/galacto-(1-->4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.     

The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved.

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 A. The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.     

Multi-site substrate binding and interplay in barley alpha-amylase 1

Certain starch hydrolases possess secondary carbohydrate binding sites outside of the active site, suggesting that multi-site substrate interactions are functionally significant. In barley alpha-amylase both Tyr380, situated on a remote non-catalytic domain, and Tyr105 in subsite -6 of the active site cleft are principal carbohydrate binding residues. The dual active site/secondary site mutants Y105A/Y380A and Y105A/Y380M show that each of Tyr380 and Tyr105 is important, albeit not essential for binding, degradation, and multiple attack on polysaccharides, while Tyr105 predominates in oligosaccharide hydrolysis. Additional delicate structure/function relationships of the secondary site are uncovered using Y380A/H395A, Y380A, and H395A AMY1 mutants.     

High-resolution crystal structures of Caldicellulosiruptor strain Rt8B.4 carbohydrate-binding module CBM27-1 and its complex with mannohexaose

Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase beta-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as beta-mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N-terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a Ka of 1 x 10(7) M(-1). Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55 angstroms and 1.06 angstoms resolution, respectively. In the crystal, CsCBM27-1 shows the typical beta-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs.     

Active site identification through geometry-based and sequence profile-based calculations: burial of catalytic clefts

Electrostatics calculations with proteins that are uniformly charged over volume can aid enzyme/non-enzyme discrimination. For known enzymes, such methods locate active sites to within 5% on the enzyme surface, in 77% of a test set. We now report that removing the dielectric boundary improves active site location to 80%, with optimal discrimination between enzymes and non-enzymes of around 80% specificity and 80% sensitivity. This calculation quantifies burial of solvent-accessible regions. Many of the true enzymes incorrectly assigned as non-enzymes have active sites at subunit boundaries. These are missed in monomer-based calculations. Catalytic and non-catalytic antibodies are studied in this context of active/binding site burial. Whilst catalytic antibodies, on average, have marginally higher active site burial than non-catalytic antibodies, these values are generally smaller than for non-antibody enzymes, possibly contributing to their relatively low turnover. Prediction of active site location improves further when sequence profile-based weights replace the uniform charge distribution, so that a combination of burial and amino acid conservation is assessed. Accuracy rises to 93% of active sites to within 5%, in the test set, for the optimal profile weights scheme. The equivalent value in a separate validation set is 89% to within 5%. Enzyme/non-enzyme and enzyme functional site predictions are made for structural genomics proteins, suggesting that a substantial majority of these are non-enzymes.     

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

Advances in molecular engineering of carbohydrate‐binding modules

Carbohydrate‐binding modules (CBMs) are non‐catalytic domains that are generally appended to carbohydrate‐active enzymes. CBMs have a broadly conserved structure that allows recognition of a notable variety of carbohydrates, in both their soluble and insoluble forms, as well as in their alpha and beta conformations and with different types of bonds or substitutions. This versatility suggests a high functional plasticity that is not yet clearly understood, in spite of the important number of studies relating protein structure and function. Several studies have explored the flexibility of these systems by changing or improving their specificity toward substrates of interest. In this review, we examine the molecular strategies used to identify CBMs with novel or improved characteristics. The impact of the spatial arrangement of the functional amino acids of CBMs is discussed in terms of unexpected new functions that are not related to the original biological roles of the enzymes. Proteins 2017; 85:1602–1617. © 2017 Wiley Periodicals, Inc.     

Circular Permutation Provides an Evolutionary Link between Two Families of Calcium-dependent Carbohydrate Binding Modules

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a β-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.     

木聚糖酶碳水化合物结合结构域研究进展

木聚糖酶含有催化活性结构域,有时还含有非催化活性结构域,促进酶与底物结合,特别是与不溶性底物的结合及降解,称为碳水化合物结合结构域(CBM),它们在木聚糖降解过程中有重要作用。以下从CBM来源,所属家族类型、对不溶性底物结合特性、与底物结合的特定氨基酸、与催化结构域间的连接肽、特别是对影响木聚糖酶稳定性的5个方面进行了综述,说明CBM对木聚糖酶性质有很大影响。自然界中碳水化合物结构复杂、难以降解,所以认识CBM相关性质对研究其与木聚糖酶的协同作用、提高木聚糖酶活性有重要意义,并根据CBM属性用于改造木聚糖酶相关性质进行了展望。     

Differential oligosaccharide recognition by evolutionarily-related beta-1,4 and beta-1,3 glucan-binding modules

Enzymes active on complex carbohydrate polymers frequently have modular structures in which a catalytic domain is appended to one or more carbohydrate-binding modules (CBMs). Although CBMs have been classified into a number of families based upon sequence, many closely related CBMs are specific for different polysaccharides. In order to provide a structural rationale for the recognition of different polysaccharides by CBMs displaying a conserved fold, we have studied the thermodynamics of binding and three-dimensional structures of the related family 4 CBMs from Cellulomonas fimi Cel9B and Thermotoga maritima Lam16A in complex with their ligands, beta-1,4 and beta-1,3 linked gluco-oligosaccharides, respectively. These two CBMs use a structurally conserved constellation of aromatic and polar amino acid side-chains that interact with sugars in two of the five binding subsites. Differences in the length and conformation of loops in non-conserved regions create binding-site topographies that complement the known solution conformations of their respective ligands. Thermodynamics interpreted in the light of structural information highlights the differential role of water in the interaction of these CBMs with their respective oligosaccharide ligands.     

Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation

Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

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 beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-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 beta1,4,-beta1,3-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.     

Two unique ligand-binding clamps of Rhizopus oryzae starch binding domain for helical structure disruption of amylose

The N-terminal starch binding domain of Rhizopus oryzae glucoamylase (RoSBD) has a high binding affinity for raw starch. RoSBD has two ligand-binding sites, each containing a ligand-binding clamp: a polyN clamp residing near binding site I is unique in that it is expressed in only three members of carbohydrate binding module family 21 (CBM21) members, and a Y32/F58 clamp located at binding site II is conserved in several CBMs. Here we characterized different roles of these sites in the binding of insoluble and soluble starches using an amylose-iodine complex assay, atomic force microscopy, isothermal titration calorimetry, site-directed mutagenesis, and structural bioinformatics. RoSBD induced the release of iodine from the amylose helical cavity and disrupted the helical structure of amylose type III, thereby significantly diminishing the thickness and length of the amylose type III fibrils. A point mutation in the critical ligand-binding residues of sites I and II, however, reduced both the binding affinity and amylose helix disruption. This is the first molecular model for structure disruption of the amylose helix by a non-hydrolytic CBM21 member. RoSBD apparently twists the helical amylose strands apart to expose more ligand surface for further SBD binding. Repeating the process triggers the relaxation and unwinding of amylose helices to generate thinner and shorter amylose fibrils, which are more susceptible to hydrolysis by glucoamylase. This model aids in understanding the natural roles of CBMs in protein-glycan interactions and contributes to potential molecular engineering of CBMs.