Carbohydrate-binding modules

This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.

• Author: ^^^Alicia Lammerts van Bueren^^^
• Responsible Curator: ^^^Al Boraston^^^ and ^^^Spencer Williams^^^

---

This page is under construction. In the meantime, please see these references for an essential introduction to the CAZy classification system: [1, 2]. CBMs, in particular, have been extensively reviewed[3, 4, 5, 6].

Overview

Figure 1. An example of modularity in a CBM-containing glycoside hydrolase. Sialidase from Micromonospora viridifaciens contains an N-terminal CBM32 (red) X20 linker (yellow) and a C-terminal catalytic GH33 module (green)[7]. Graphical representation of modularity in amino acid sequence (top) and 3D crystal structure (bottom) PDB ID 1eut.

Carbohydrate-binding modules (CBMs) are defined as a stretch of amino acid sequence within a larger encoded protein sequence and fold into a discreet and independent module, forming part of a larger multi-modular protein (Figure 1). The role of a CBM is to bind to carbohydrate ligand and direct the catalytic machinery onto its substrate, thus enhancing the catalytic efficiency of the multimodular carbohydrate-active enzyme. CBMs are themselves devoid of any catalytic activity. CBMs are most commonly associated with Glycoside Hydrolases but have also been identified in Polysaccharide Lyases, polysaccharide oxidases, Glycosyltransferases and plant cell wall-binding expansins [8].

CBMs themselves do not undergo any conformational changes when binding ligand. Rather, the topography of the carbohydrate-binding site is preformed to be complementary to the shape of the target ligand (see Types). This is achieved by the presence of amino acid side chains and loops within the CBM binding pocket. However multimodular enzymes as a whole may be quite flexible and experience complete conformational changes when binding substrate. Flexible loop regions between adjacent modules can allow for shifts in the orientation and direction of the catalytic module with respect to the CBM on the target substrate [9].

History of CBMs

CBMs were initially characterized as cellulose binding domains (CBDs) in cellobiohydrolases CBHI and CBHII from Trichoderma reesei [10, 11] and cellulases CenA and CexA from Cellulomonas fimi [12]. Limited proteolysis experiments of these enzymes yielded truncated enzyme products that showed a reduced or complete loss in their ability to hydrolyze cellulose substrates. The reduction in enzymatic activity was attributed to the loss of ~100 amino acid C-terminal domains which prevented the adsorbption of the enzymes onto cellulose substrate. Thus it was proposed that these independent "domains" are critical for targeting the enzymes onto its substrate and enhancing their hydrolytic activity.

CBDs were previously categorized into 13 Types based on amino acid sequence similarities [13]. This classification system became complicated when similar functional domains from non-cellulolytic carbohydrate-active enzymes were discovered that did not bind cellulose but met all of the criteria of a CBD. The term carbohydrate-binding module was proposed to solve this problem to be inclusive of all ancillary modules with non-catalytic carbohydrate-binding function (for a review see [3]).

Classification

Sequence Based Classification

Carbohydrate-binding modules are currently classified into 67 families based on amino acid sequence similarities (May 2013), which are available through the Carbohydrate Active enZyme database. Sequence-based relationships often cluster together modules with similar structural folds and carbohydrate-binding function. While this is true for most CBM families, there are several families that exhibit diversity in the carbohydrate ligands they target (examples include CBM6, CBM32)

Fold

Structural information for 54 of the 67 CBM families is known. The most common fold exhibited by CBMs is the beta-sandwich fold which is comprised of two overlapping beta-sheets consisting of 3 - 6 antiparallel beta strands (Figure 2). The ligand binding site is located primarily on the same face of a beta-sheet (Figure 2A), but may also be positioned on the edge of the beta-sheet within the joining loop region (Figure 2B). There are examples of CBMs exhibiting dual binding sites [14]. Other folds include the beta-trefoil fold, cysteine knot, OB fold, the hevein and hevein-like and unique folds [3].

Figure 2. Classical CBM beta-sandwich fold. C-terminal family CBM27 from Thermotoga maritima mannanase (A)(side and front view, PDB ID 1OF4) [15] and C-terminal family CBM6 from Clostridium stercorarium xylanase (B) (PDB ID 1NAE) [16] showing binding sites on the face (A) and edge (B) of the beta sandwich fold respectively.

Types

CBMs are classified into three main Types defined by the shape and degree of polymerization of their target ligand. A recent review has modified the classification of CBM Types to be as follows [17]:

• Type A: bind to crystalline surfaces of cellulose and chitin (example families CBM1, CBM2, CBM3, CBM5, CBM10). Their binding sites are planar and rich in aromatic amino acid residues creating a flat platform to bind to the flat polycrystalline chitin/cellulose surface. Type A CBMs are unique and differ significantly from Type B or C.
• Type B: bind internal glycan chains (endo-type). The most abundant CBM type. Type B binding sites appear as extended grooves or clefts comprised of binding subsites to accommodate longer sugar chains with 4 or more monosaccharide units.
• Type C: bind termini of glycans (reducing/non-reducing ends, exo-type). Type C binding sites are short pockets for recognizing short sugar ligands containing 1 - 3 monosaccharide units (example families CBM9, CBM13, CBM47). Families containing Type C CBMs often include lectins as members.

Properties of Carbohydrate Binding Interactions

Functional Roles of CBMs

CBMs carry out four main functional roles:

Targeting Effect: CBMs target the enzyme to distinct regions within a larger macromolecular polysaccharide substrate (reducing, non-reducing ends, internal surface structures).

Proximity Effect: CBMs increase the concentration of enzyme in close proximity to its polysaccharide substrate. This leads to more rapid and efficient substrate degradation.

Disruptive Effect: Disruptive role has previously been described for cellulose binding CBM2a. Additionally starch binding CBM20 may have a disruptive role in amylose while dual-associated CBM41 modules may have a disruptive role in degrading glycogen granules. CBM33 was thought to have a disruptive effect on chitin, however these have now been reclassified as Copper-dependent lytic polysaccharide monooxygenases [18] and are reclassified as Auxillary Activity Family 9.

Adhesion: CBMs have been shown to adhere enzymes onto the surface of bacterial cell wall components while exhibiting catalytic activity on an external neighboring carbohydrate substrate. For example, CBM35 modules have been shown to interact with the surface glucuronic acid containing sugars in the cell wall of Amycolatopsis orientalis while the catalytic module is active on external chitosan likely originating from the cell wall of competing soil fungal species [19].

Driving Forces of CBM/Carbohydrate Interactions

There are two key features that drive CBM/carbohydrate interactions. Extensive hydrogen bonding occurs between the hydroxyl groups of carbohydrate ligands and polar amino acid residues within the binding site. Additional water-mediated hydrogen bonding networks between these groups can also be found in the binding site. By far the most important characteristic driving force mediating protein-carbohydrate interactions is the position and orientation of aromatic amino acid residues (Try, Tyr and sometimes Phe) within the binding site. These essential planar residues form hydrophobic stacking interactions with the planar face of sugar rings. Weak intermolecular electrostatic interactions occur between C-H and pi electrons in the planar ring systems and contribute 1.5 - 2.5 kcal/mol energy to the binding reaction [20].

CBM Promiscuity

Because of the diversity of carbohydrate structures and motifs found in plant and mammalian glycans, some CBMs feature promiscuity in ligand recognition. While the core monosaccharide in the primary subsite remains important for initial recognition of carbohydrate ligand, CBMs may exhibit flexibility in what sugar monomers can be accommodated in binding subsites. Examples include...

Criteria for Defining a new CBM family

In order to define a new CBM family, one must:

1. Demonstrate an independent module as part of a larger carbohydrate-active enzyme.
2. Demonstrate binding to carbohydrate ligand.
3. Additional family members are then determined based on amino acid sequence similarity. To be defined as a true CBM, it must form part of a larger amino acid sequence encoding a putative CAZyme (or enzyme with demonstrated activity on a carbohydrate-containing substrate and the CBM enhances the catalytic efficiency of the enzyme by binding with or in close proximity of the substrate).

Amino acid sequence-based classification of a CBM family may lead to the incorporation of other carbohydrate binding proteins within a given family, including lectins (such as ricin (CBM13), tachycitin (CBM14), wheat germ agglutinin (CBM18), fucolectin (CBM47), and malectin (CBM57)) and periplasmic solute binding proteins (such as CBM32). The community is open to incorporation of all carbohydrate-binding proteins within the CBM classification system based on the above criteria.

Studying CBM-ligand Interactions

Laboratory approaches to studying the binding function of carbohydrate-binding modules has been extensively reviewed by Abbott and Boraston [21]. Typically, molecular biology techniques are used to overproduce a CBM protein in a host strain such as Escherichia coli which is then isolated and purified. Initial screening for carbohydrate binding interactions can be performed using screening techniques such as microarrays [22] or fluorescence microscopy techniques [22] [23] [24]. Several approaches can be taken to verify and quantify CBM-polysaccharide interaction, including affinity gel electrophoresis, UV difference and fluorescence spectroscopy, solid state depletion assay and isothermal titration calorimetry [25].

Overall demonstration of carbohydrate binding function by CBMs is essential to understanding how these associated modules confer enzymatic specificity to carbohydrate-active enzymes. Features of CBMs are currently being exploited to create designer CAZymes with enhanced or modified carbohydrate recognition functions [26].

References

1. Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). DOI: 10.1042/BJ20080382

2. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
3. Boraston AB, Bolam DN, Gilbert HJ, and Davies GJ. (2004). Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J. 2004;382(Pt 3):769-81. DOI:10.1042/BJ20040892 |
4. Hashimoto H (2006). Recent structural studies of carbohydrate-binding modules. Cell Mol Life Sci. 2006;63(24):2954-67. DOI:10.1007/s00018-006-6195-3 |
5. Shoseyov O, Shani Z, and Levy I. (2006). Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol Rev. 2006;70(2):283-95. DOI:10.1128/MMBR.00028-05 |
6. Guillén D, Sánchez S, and Rodríguez-Sanoja R. (2010). Carbohydrate-binding domains: multiplicity of biological roles. Appl Microbiol Biotechnol. 2010;85(5):1241-9. DOI:10.1007/s00253-009-2331-y |
7. Gaskell A, Crennell S, and Taylor G. (1995). The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure. 1995;3(11):1197-205. DOI:10.1016/s0969-2126(01)00255-6 |
8. Georgelis N, Tabuchi A, Nikolaidis N, and Cosgrove DJ. (2011). Structure-function analysis of the bacterial expansin EXLX1. J Biol Chem. 2011;286(19):16814-23. DOI:10.1074/jbc.M111.225037 |
9. Ficko-Blean E, Gregg KJ, Adams JJ, Hehemann JH, Czjzek M, Smith SP, and Boraston AB. (2009). Portrait of an enzyme, a complete structural analysis of a multimodular {beta}-N-acetylglucosaminidase from Clostridium perfringens. J Biol Chem. 2009;284(15):9876-84. DOI:10.1074/jbc.M808954200 |

10. Van Tilbeurgh, H., Tomme P., Claeyssens M., Bhikhabhai R., Pettersson G.(1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS Lett. 204,223–227. [1]

11. Tomme P, Van Tilbeurgh H, Pettersson G, Van Damme J, Vandekerckhove J, Knowles J, Teeri T, and Claeyssens M. (1988). Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988;170(3):575-81. DOI:10.1111/j.1432-1033.1988.tb13736.x |
12. Gilkes NR, Warren RA, Miller RC Jr, and Kilburn DG. (1988). Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988;263(21):10401-7. | Google Books | Open Library

13. Tomme, P., Warren, R.A., Miller, R.C., Jr., Kilburn, D.G. & Gilkes, N.R. (1995) in Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J.N. & Penner, M., eds.), Cellulose-binding domains: classification and properties. pp. 142-163, American Chemical Society, Washington.

14. Pires VM, Henshaw JL, Prates JA, Bolam DN, Ferreira LM, Fontes CM, Henrissat B, Planas A, Gilbert HJ, and Czjzek M. (2004). 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. J Biol Chem. 2004;279(20):21560-8. DOI:10.1074/jbc.M401599200 |
15. Boraston AB, Revett TJ, Boraston CM, Nurizzo D, and Davies GJ. (2003). Structural and thermodynamic dissection of specific mannan recognition by a carbohydrate binding module, TmCBM27. Structure. 2003;11(6):665-75. DOI:10.1016/s0969-2126(03)00100-x |
16. Boraston AB, Notenboom V, Warren RA, Kilburn DG, Rose DR, and Davies G. (2003). Structure and ligand binding of carbohydrate-binding module CsCBM6-3 reveals similarities with fucose-specific lectins and "galactose-binding" domains. J Mol Biol. 2003;327(3):659-69. DOI:10.1016/s0022-2836(03)00152-9 |
17. Gilbert HJ, Knox JP, and Boraston AB. (2013). Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Curr Opin Struct Biol. 2013;23(5):669-77. DOI:10.1016/j.sbi.2013.05.005 |
18. Vaaje-Kolstad G, Westereng B, Horn SJ, Liu Z, Zhai H, Sørlie M, and Eijsink VG. (2010). An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science. 2010;330(6001):219-22. DOI:10.1126/science.1192231 |
19. Montanier C, van Bueren AL, Dumon C, Flint JE, Correia MA, Prates JA, Firbank SJ, Lewis RJ, Grondin GG, Ghinet MG, Gloster TM, Herve C, Knox JP, Talbot BG, Turkenburg JP, Kerovuo J, Brzezinski R, Fontes CM, Davies GJ, Boraston AB, and Gilbert HJ. (2009). Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function. Proc Natl Acad Sci U S A. 2009;106(9):3065-70. DOI:10.1073/pnas.0808972106 |
20. Meyer EA, Castellano RK, and Diederich F. (2003). Interactions with aromatic rings in chemical and biological recognition. Angew Chem Int Ed Engl. 2003;42(11):1210-50. DOI:10.1002/anie.200390319 |
21. Abbott DW and Boraston AB. (2012). Quantitative approaches to the analysis of carbohydrate-binding module function. Methods Enzymol. 2012;510:211-31. DOI:10.1016/B978-0-12-415931-0.00011-2 |
22. van Bueren AL, Higgins M, Wang D, Burke RD, and Boraston AB. (2007). Identification and structural basis of binding to host lung glycogen by streptococcal virulence factors. Nat Struct Mol Biol. 2007;14(1):76-84. DOI:10.1038/nsmb1187 |
23. McCartney L, Blake AW, Flint J, Bolam DN, Boraston AB, Gilbert HJ, and Knox JP. (2006). Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules. Proc Natl Acad Sci U S A. 2006;103(12):4765-70. DOI:10.1073/pnas.0508887103 |
24. Hervé C, Rogowski A, Blake AW, Marcus SE, Gilbert HJ, and Knox JP. (2010). Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects. Proc Natl Acad Sci U S A. 2010;107(34):15293-8. DOI:10.1073/pnas.1005732107 |
25. Lammerts van Bueren A and Boraston AB. (2004). Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites. J Mol Biol. 2004;340(4):869-79. DOI:10.1016/j.jmb.2004.05.038 |
26. Cuskin F, Flint JE, Gloster TM, Morland C, Baslé A, Henrissat B, Coutinho PM, Strazzulli A, Solovyova AS, Davies GJ, and Gilbert HJ. (2012). How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity. Proc Natl Acad Sci U S A. 2012;109(51):20889-94. DOI:10.1073/pnas.1212034109 |
27. Adams JJ, Gregg K, Bayer EA, Boraston AB, and Smith SP. (2008). Structural basis of Clostridium perfringens toxin complex formation. Proc Natl Acad Sci U S A. 2008;105(34):12194-9. DOI:10.1073/pnas.0803154105 |
28. Boraston AB, Ficko-Blean E, and Healey M. (2007). Carbohydrate recognition by a large sialidase toxin from Clostridium perfringens. Biochemistry. 2007;46(40):11352-60. DOI:10.1021/bi701317g |
29. Lammerts van Bueren A, Ficko-Blean E, Pluvinage B, Hehemann JH, Higgins MA, Deng L, Ogunniyi AD, Stroeher UH, El Warry N, Burke RD, Czjzek M, Paton JC, Vocadlo DJ, and Boraston AB. (2011). The conformation and function of a multimodular glycogen-degrading pneumococcal virulence factor. Structure. 2011;19(5):640-51. DOI:10.1016/j.str.2011.03.001 |