The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Abstract

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 Å. The protein is a ␤-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.

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…A similar hierarchical classification exists for glycosyltransferases, with both the family and clan groupings predictive of functional and structural features of the members [39,40]. Although fold similarities between CBMs have been demonstrated, and the existence of superfamilies has been suggested [41,42], there are currently no formal 'super' groupings of the 39 CBM families. To approach this issue, we manually classified the structures from 22 different CBM families into seven 'fold families' (Table 2; Figure 1).…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…In some of the 59 CBM families, exemplified by CBM1, CBM10, and CBM20, ligand specificity is invariant (Linder and Teeri, 1997;Southall et al, 1999;Raghothama et al, 2000), while in some families, such as CBM6 (Czjzek et al, 2001;Pires et al, 2004), CBM4 (Boraston et al, 2002b), and CBM35 (Tunnicliffe et al, 2005;Montanier et al, 2009b), carbohydrate recognition is highly variable. In addition to defining a phylogenetic relationship between CBMs by clustering these modules into sequence-based families, they have also been classified into three categories (types A, B, and C) based on the topology of their ligand-binding sites and their mode of ligand recognition (for review, see Boraston et al, 2004; Fig.…”

“…While the structures of CEs, PLs, and CBMs are dominated by the a/b-hydrolase (Correia et al, 2008), parallel b-helix (Pickersgill et al, 1994), and jelly roll (or b-sandwich; Czjzek et al, 2001) folds, respectively, there are a large number of different folds within the GHs, which are discussed below. Indeed, the same criteria used to include enzymes in the same GH have now been used to cluster a proportion of the GHs into 14 different clans (Cantarel et al, 2009).…”

“…While the perpendicular arrangement of aromatic residues in xylan-binding CBMs is a common feature, modules that recognize the hemicellulosic polymer can adopt different ligand-binding strategies. Thus, in CBM4, CBM6, and CBM22, xylan recognition is dominated by a single Xyl residue that is sandwiched between a pair of planar aromatic residues within a deep ligand-binding cleft (Czjzek et al, 2001;Charnock et al, 2002a;Simpson et al, 2002). While this binding mode confers higher affinity for isolated xylan chains, CBMs that recognize xylan through the asymmetric distribution of aromatic residues display more versatile ligand recognition; they are able to bind to the hemicellulose within in a variety of terrestrial plant cell walls, a specificity that is not displayed by the modules from CBM4, CBM6, and CBM22 (McCartney et al, 2006).…”

“…The vast majority of these modules display a jelly roll fold comprising two antiparallel b-sheets that form the two surfaces of these proteins. Ligand binding occurs on the concave surface presented by one of the b-sheets (Boraston et al, 2002b) or in the loops that connect the two b-sheets (defined hereafter as site 1; Czjzek et al, 2001;Montanier et al, 2009b).…”