Carbohydrate Binding Module Family 3

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• Author: ^^^Harry Gilbert^^^ and ^^^Ed Bayer^^^
• Responsible Curator: ^^^Ed Bayer^^^

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CAZy DB link

https://www.cazy.org/CBM3.html

Ligand specificities

CBM3 is a Gram-positive bacterial family of protein modules that comprise around 150 amino acids. The family is divided into four subgroups, CBM3a-d. The major ligand recognised by CBM3as and CBM3bs is crystalline cellulose with an affinity (K_D) of 0.4 µM determined by depletion isotherms [1]. Isothermal titration calorimetry showed that binding to crystalline cellulose was entropically driven consistent with apolar interactions resulting in the release of caged water molecules from a ligand with a restricted conformation [2]. CBM3s that bind to crystalline cellulose also interact with chitin and xyloglucan with an affinity ~500 lower than for crystalline cellulose.

Interaction with soluble xyloglucans by CBM3s was enthalpically driven with changes in entropy having a negative impact on affinity [2]. The site of binding of a CBM3 from the Clostridium thermocellulum scaffoldin (CipA) to crystalline cellulose was determined by transmission electron microscopy with detection of the protein by immuno-gold labelling. The data showed that the CBM3 bound to the 110 face of Valonia cellulose [3]. The binding profile and site of cellulose recognition show that CBM3s are type A modules. The three CBM3s from anti-sigma sensors displayed different specificities; Cthe_0059 CBM3b bound to a range of plant cell wall polysaccharides (PCWPs), Cthe_0404 CBM3b interacted weakly to xyloglucan but not to any other PCWP, and Cthe_0267 CBM3 bound primarily to crystalline and amorphous cellulose [4, 5].

Structural Features

Figure 1. The fold of the cellulose binding CBM3a from the Clostridium thermocellum scaffoldin CipA (PDB ID 1NBC), highlighting the planar ligand binding site comprising five residues. The structure is rotated 90 degrees to illustrate the location of these residues on the β-sheet.

The crystal structure of CBM3 from the C. thermocellum scaffoldin CipA revealed a classical β-jelly-roll fold consisting of nine β-strands in two antiparallel β-sheets comprising four (1, 2, 7, 4; β-sheet 1) and five (9, 8, 3, 6, 5; β-sheet 2) β strands, respectively [6] (PDB ID 1NBC). β-sheet 1 forms a flat surface that contains a linear array of five residues that presents a planar hydrophobic surface comprising a His, Trp, Tyr and an Arg-Asp ion pair (Figure 1). The residues in the planar strip were predicted to make apolar interactions with glucose molecules n, n+1, n+3 and n+5, consistent with mutagenesis data showing that each of the five amino acids played an important role in binding cellulose [7]. Structures of CBMbs from other cellulosome-producing species followed that reinforced the original structural findings [8, 9, 10]. In other CBM3 modules that bind to cellulose, such as in the anti-σ-cell surface sensor RsgI1 (Cthe_0059), the His and ion pair are replaced by a Tyr and Phe, thus the hydrophobic planar binding site comprises four aromatic amino acids [5]. In a second cellulose-binding CBM3 located in an Rsgl sensor (Rsgl2, Cthe_0267), the aromatic planar strip is truncated, but lies planar with a hydrophobic protruding loop that is predicted to contribute to the cellulose binding site of the protein, similar to a group d CBM3 present in a GH48 exo-cellulase [11]. In addition to the hydrophobic strips it has also been proposed that highly conserved polar residues may be able to make productive hydrogen bonds with two additional cellulose chains in the microfibril [5, 6].

In contrast to the planar face presented by β-sheet 1, β-sheet 2 displays a concave surface or shallow groove that contains highly conserved aromatic residues [6], suggesting that these hydrophobic amino acids are functionally significant. It has been proposed that the shallow cleft is involved in binding Pro-Thr linker segments and thus may contribute to structural organization of these multimodular proteins [12].

Functionalities

CBM3s are derived from the scaffoldins [13] (non-catalytic proteins that that play an integral role in the assembly of multienzyme plant cell wall degrading complexes termed cellulosomes (see [14] for review), sensor proteins that detect cellulose [4] and a range of cellulases (e.g. [15, 16, 17]). In general CBM3s are separated from the other modules in these proteins by Pro-Thr-rich linker sequences. In some instances, however, group c CBM3 members (CBM3cs) are integral components of the substrate binding cleft of GH9 cellulases (e.g. [16, 17, 18, 19]) In these enzymes the CBM3c modules, as discrete entities, do not bind to cellulose (reflecting the lack of conserved ligand binding residues) but play a pivotal role in the capacity of the cellulases to attack crystalline forms of the polysaccharide [17]. It was proposed that the replacement of aromatic residues with conserved polar amino acids, altered the function of CBM3cs from an anchoring role. In the model proposed the polar residues in CBM3s replace the inter-chain hydrogen bonds within crystalline cellulose. The resultant disruption of the crystalline polysaccharide releases the cellulose chain on the centre of the CBM3c, which could then be fed into the active site cleft of the catalytic domain. Several studies have shown that CBM3 modules have enhanced the activity of cellulases [20] and a range of other plant cell wall degrading enzymes [21, 22]. These modules have also been used to probe the structure of plant cell walls [23, 24].

Family Firsts

First Identified

The first CBM3 to be identified (CipA-CBM3) was from the C. thermocellum scaffoldin CipA [13].

First Structural Characterization

The first crystal structure of a CBM3, indeed of any CBM, is CipA-CBM3 [6]

References

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7. Benhar, I., Tamarkin, A., Marash, L., Berdichevsky, Y., Yaron, S., Shoham, Y., Lamed, R., and Bayer, E. A. (2001) Phage display of cellulose binding domains for biotechnological application. In Glycosyl Hydrolases for Biomass Conversion (Himmel, M. E., Baker, J. O., and Saddler, J. N., Eds.), pp 168-189, American Chemical Society, Washington, DC. DOI:10.1021/bk-2001-0769.ch010.