Structure of a Mannan-specific Family 35 Carbohydrate- Binding Module: Evidence for Significant Conformational Changes upon Ligand Binding Richard B. Tunnicliffe 1 , David N. Bolam 2 , Gavin Pell 2 , Harry J. Gilbert 2 and Mike P. Williamson 1 * 1 Department of Molecular Biology and Biotechnology University of Sheffield, Western Bank, Sheffield S10 2TN, UK 2 School of Cell and Molecular Biosciences, University of Newcastle upon Tyne Newcastle upon Tyne NE1 7RU UK 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 b-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 b-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 b-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 accommo- dation 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. q 2005 Elsevier Ltd. All rights reserved. Keywords: carbohydrate-binding module; NMR structure; mannan; binding specificity; decorated oligosaccharides *Corresponding author Introduction The interaction of proteins with specific carbo- hydrates is widespread throughout nature and is central to many biological processes. Plant struc- tural polysaccharides constitute the most abundant source of renewable carbon in the biosphere, and thus degradation of these polymers is essential for the continual recycling of carbon. The microbial glycoside hydrolases, lyases and esterases that mediate this task are typically modular proteins that contain one or more non-catalytic, carbo- hydrate-binding modules (CBMs) in addition to the catalytic module. CBMs act by binding to regions of the complex carbohydrate network of plant cell-walls, and recruit the attached catalytic module onto the surface of the substrate, increasing its effective concentration, which leads to enhanced catalysis. 1,2 In addition, it has been suggested that CBMs can disrupt the inter-chain interactions of the polysaccharide matrix, thereby making it more accessible for hydrolysis, although this mechanism of action has been observed only in a starch-binding module 3 and a single cellulose-binding module. 4 CBMs have been grouped into families on the basis of sequence similarities†; 5 the number of families has grown rapidly to 42 and continues to expand. Currently, representative structures from over half of the families are available, the most abundant fold being the b-jelly-roll, members of which form a superfamily. 6 CBM structures can be grouped into three broad classes on the basis of the 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: CBM, carbohydrate-binding module; DiGalMan, 6 3 ,6 4 -a-D-galactosyl-mannopentaose; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy. E-mail address of the corresponding author: m.williamson@sheffield.ac.uk † afmb.cnrs-mrs.fr/CAZY doi:10.1016/j.jmb.2005.01.038 J. Mol. Biol. (2005) 347, 287–296