Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.     

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.     

The crystal structure of the family 6 carbohydrate binding module from Cellvibrio mixtus endoglucanase 5a in complex with oligosaccharides reveals two distinct binding sites with different ligand specificities

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain non-catalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis. Family 6 CBMs (CBM6) are highly unusual because they contain two distinct clefts (cleft A and cleft B) that potentially can function as binding sites. Henshaw et al. (Henshaw, J., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes, C. M. G. A., and Gilbert, H. J. (2003) J. Biol. Chem. 279, 21552-21559) show that CmCBM6 contains two binding sites that display both similarities and differences in their ligand specificity. Here we report the crystal structure of CmCBM6 in complex with a variety of ligands that reveals the structural basis for the ligand specificity displayed by this protein. In cleft A the two faces of the terminal sugars of beta-linked oligosaccharides stack against Trp-92 and Tyr-33, whereas the rest of the binding cleft is blocked by Glu-20 and Thr-23, residues that are not present in CBM6 proteins that bind to the internal regions of polysaccharides in cleft A. Cleft B is solvent-exposed and, therefore, able to bind ligands because the loop, which occludes this region in other CBM6 proteins, is much shorter and flexible (lacks a conserved proline) in CmCBM6. Subsites 2 and 3 of cleft B accommodate cellobiose (Glc-beta-1,4-Glc), subsite 4 will bind only to a beta-1,3-linked glucose, whereas subsite 1 can interact with either a beta-1,3- or beta-1,4-linked glucose. These different specificities of the subsites explain how cleft B can accommodate beta-1,4-beta-1,3- or beta-1,3-beta-1,4-linked gluco-configured ligands.     

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.     

Glycoside hydrolase carbohydrate-binding modules as molecular probes for the analysis of plant cell wall polymers

Novel molecular probes have been developed for the analysis and detection of polysaccharides in plant cell walls using carbohydrate-binding modules (CBMs) derived from modular glycoside hydrolases belonging to families 2a, 6, and 29. Recombinant forms of these proteins containing his-tags, in conjunction with anti-his-tag detection, provide a flexible system that utilizes CBMs as molecular probes in a range of applications. Assays for the rapid analysis of the binding of CBMs to polysaccharides and oligosaccharides using nitrocellulose-based CBM macroarrays and microtiter plate-based CBM capture and competitive-inhibition assays are described. We also demonstrate the use of CBMs with his-tags for the localization of their target ligands in planta. The generation of molecular probes from other families of CBMs will dramatically increase the repertoire of molecular probes available to determine the developmental and functional aspects of plant cell walls.     

Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Thermal unfolding and modular architecture of Clostridium stercorarium Xyn10B

To examine the possibility of module interaction in the thermal unfolding of different modular architectures, four truncated proteins were constructed from Clostridium stercorarium Xyn10B: a family 10 catalytic module (CM10), a polypeptide compound of one family 22 carbohydrate-binding module (CBM22-2) and the catalytic module (CBM22-CM10), two family 22 CBMs and the catalytic module (2CBM22-CM10), and only two family 22 CBMs (2CBM22). Thermal unfolding of four proteins were observed by differential scanning calorimetry. CM10 was unfolded reversibly and denatured as one component. The unfolding of protein CBM22-CM10 comprising CBM22-2 connected with CM10 was irreversible, and can be assumed to be one-component denaturation. Protein 2CBM22, with two CBM22s in tandem, unfolded as two independent modules. However, 2CBM22-CM10, with two CBM22s, unfolded as two and not the expected three separate components. These findings constitute the first reported case in which differences in thermal unfolding units and mechanisms were derived from differences in the modular architectures of proteins.     

The Overall Architecture and Receptor Binding of Pneumococcal Carbohydrate-Antigen-Hydrolyzing Enzymes

The TIGR4 and SP3-BS71 strains of Streptococcus pneumoniae each produce family 98 glycoside hydrolases, called Sp4GH98 and Sp3GH98, respectively, which have different modular architectures and substrate specificities. Sp4GH98 degrades the Lewis^Y antigen and possesses three C-terminal family 47 carbohydrate-binding modules (CBMs) that bind to this substrate. Sp3GH98 degrades the blood group A/B antigens and has two N-terminal family 51 CBMs that are of unknown function. Here, we examine the complex carbohydrate-binding specificity of the family 51 CBMs from Sp3GH98 (referred to as CBM51-1 and CBM51-2), the structural basis of this interaction, and the overall solution conformations of both Sp3GH98 and Sp4GH98, which are shown to be fully secreted proteins. Through glycan microarray binding analysis and isothermal titration calorimetry, CBM51-1 is found to bind specifically to the blood group A/B antigens. However, due to a series of relatively small structural rearrangements that were revealed in structures determined by X-ray crystallography, CBM51-2 appears to be incapable of binding carbohydrates. Analysis of small-angle X-ray scattering data in combination with the available high-resolution X-ray crystal structures of the Sp3GH98 and Sp4GH98 catalytic modules and their CBMs yielded models of the biological solution structures of the full-length enzymes. These studies reveal the complex architectures of the two enzymes and suggest that carbohydrate recognition by the CBMs and the activity of the catalytic modules are not directly coupled.     

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.     

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.     

Xyloglucan is recognized by carbohydrate-binding modules that interact with beta-glucan chains

Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of beta-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a beta-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated beta-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains.     

Thermal Unfolding and Modular Architecture of Clostridium stercorarium Xyn10B

To examine the possibility of module interaction in the thermal unfolding of different modular architectures, four truncated proteins were constructed from Clostridium stercorarium Xyn10B: a family 10 catalytic module (CM10), a polypeptide compound of one family 22 carbohydrate-binding module (CBM22-2) and the catalytic module (CBM22-CM10), two family 22 CBMs and the catalytic module (2CBM22-CM10), and only two family 22 CBMs (2CBM22). Thermal unfolding of four proteins were observed by differential scanning calorimetry. CM10 was unfolded reversibly and denatured as one component. The unfolding of protein CBM22-CM10 comprising CBM22-2 connected with CM10 was irreversible, and can be assumed to be one-component denaturation. Protein 2CBM22, with two CBM22s in tandem, unfolded as two independent modules. However, 2CBM22-CM10, with two CBM22s, unfolded as two and not the expected three separate components. These findings constitute the first reported case in which differences in thermal unfolding units and mechanisms were derived from differences in the modular architectures of proteins.     

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.     

Function of the family-9 and family-22 carbohydrate-binding modules in a modular beta-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B

Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.     

Functions of family-22 carbohydrate-binding modules in Clostridium josui Xyn10A

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.     

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.     

Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding

Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus beta-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical beta-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two beta-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.     

X-ray crystal structure of a non-crystalline cellulose-specific carbohydrate-binding module: CBM28

Natural cellulose exists as a composite of different forms, which have historically been broadly characterized as "crystalline" or "amorphous". The recognition of both of these forms of cellulose by the carbohydrate-binding modules (CBM) of microbial glycoside hydrolases is central to natural and efficient biotechnological conversion of plant cell wall biomass. There is increasing evidence that, at least some, individual binding modules target distinct and different regions of non-crystalline "amorphous" cellulose. Competition experiments show that CBM28 modules do not compete with CBM17 modules when binding to non-crystalline cellulose. The structure of the BspCBM28 (http://afmb.cnrs-mrs.fr/CAZY/) module from the Bacillus sp. 1139 family GH5 endoglucanase, comprising a 191 amino acid protein, has therefore been determined at 1.4A resolution using single isomorphous replacement with anomalous scattering methods. The structure reveals a "beta-jelly roll" topology, with high degree of similarity to the structure of CBM17 domains. Sequence and structural conservation strongly suggests that these two families of domains have evolved through gene duplication and subsequent divergence. The ligand-binding site "topographies" of CBMs from families 28, 17 and 4 begins to shed light on the differential recognition of non-crystalline cellulose by multi-modular plant cell wall-degrading enzymes.     

The structural basis for the ligand specificity of family 2 carbohydrate-binding modules

The interactions of proteins with polysaccharides play a key role in the microbial hydrolysis of cellulose and xylan, the most abundant organic molecules in the biosphere, and are thus pivotal to the recycling of photosynthetically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). The largest prokaryotic CBM family, CBM2, contains members that bind cellulose (CBM2a) and xylan (CBM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate interaction is rotated by 90 degrees compared with its position in CBM2a (thus matching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Arg-262-->Gly), a CBM2b xylan-binding module completely loses its affinity for xylan and becomes a cellulose-binding module. The structural effect of the mutation has been revealed using NMR spectroscopy, which confirms that Trp-259 rotates 90 degrees to lie flat against the protein surface. Except for this one residue, the mutation only results in minor changes to the structure. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the recognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.