Binding Specificity and Thermodynamics of a Family 9 Carbohydrate-Binding Module from Thermotoga maritima Xylanase 10A † Alisdair B. Boraston, ‡,§,| A. Louise Creagh, ‡,|,⊥ Md. Mustafa Alam, ‡,§,# Jeffery M. Kormos, | Peter Tomme, ‡,§,|,O Charles A. Haynes, ‡,|,⊥ R. Antony J. Warren, ‡,§ and Douglas G. Kilburn* ,‡,§,| Protein Engineering Network of Centres of Excellence, Department of Microbiology and Immunology, Department of Chemical and Biological Engineering, and The Biotechnology Laboratory, UniVersity of British Columbia, VancouVer, British Columbia, V6T 1Z3 Canada ReceiVed January 26, 2001 ABSTRACT: The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K a ) for adsorption to insoluble polysaccharides are 1 × 10 5 to 3 × 10 5 M -1 . Of the soluble polysaccharides tested, CBM9-2 binds to barley -glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10 3 M -1 for monosaccharides to 10 6 M -1 for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes. Many organisms have evolved diverse polysaccharolytic enzyme systems to tap the abundant carbohydrate energy source found in plant biomass. A large proportion of these enzymes have modular structures comprising catalytic mod- ules, carbohydrate-binding modules (CBMs), 1 modules that mediate protein-protein interactions, modules of unknown function, and modules that appear to serve only as linkers between modules (1). CBMs, previously called cellulose- binding domains (CBDs), are grouped into families of related amino acid sequences, of which there are 13 at present (2, 3). CBMs within families and from different families can have different binding specificities. The N-terminal family 4 CBM, CBM4-1, of endoglucanase Cel9B from the mesophilic bacterium Cellulomonas fimi, binds to cellooligosaccharides and to amorphous cellulose but not to crystalline cellulose (4, 5). The family 2a CBM, CBM2a, of xylanase 10A from C. fimi, binds irreversibly to crystalline cellulose and to crystalline regions of amorphous cellulose preparations but not to soluble cellulose or cellooligosaccharides (6-9). Thermodynamic studies by microcalorimetry show that the binding mechanisms of CBM4-1 and CBM2a are distinctly different. The binding of CBM4-1 is dominated by a favorable change in enthalpy, which is partly compensated by unfavorable entropy changes. Binding is also characterized by a small negative change in heat capacity (4). This indicates that the interaction between CBM4-1 and cellooligosaccha- rides involves both H-bonding and van der Waals interac- tions. The solution structure of CBM4-1 (10) shows that its binding site is a groove lined with hydrophobic amino acid residues and flanked with planar polar amino acid residues, suggesting an interaction with cellulose consistent with the calorimetry results. In contrast, the interaction of CBM2a with cellulose is dominated by a favorable change in entropy, a favorable, but very small, change in enthalpy, and a large negative change in heat capacity (8). The entropic driving force was interpreted to arise from ordered water molecules returned to the bulk solution by the dehydration of the polypeptide and the cellulose surface. The hydrophobic nature of the CBM2a binding site supports this (9, 11), indicating that thermodynamic analysis of CBM-ligand interactions thus provides some understanding of the mech- anisms as well as the energetics of binding. However, such a study has not yet been applied to CBMs from hyperther- mophilic sources, which may have evolved novel charac- teristics to operate at elevated temperatures. † This work was supported by grants from the Protein Engineering Network of Centres of Excellence, CBD Technologies Inc., and the Natural Sciences and Engineering Research Council of Canada (D.G.K.). * To whom correspondence should be addressed: The Biotechnology Laboratory, University of British Columbia, 237-6174 University Blvd., Vancouver, BC, Canada V6T 1Z3. Tel: (604) 822-4182. Fax: (604) 822-2114. E-mail: kilburn@interchange.ubc.ca. ‡ Protein Engineering Network of Centres of Excellence. § Department of Microbiology and Immunology. | The Biotechnology Laboratory. ⊥ Department of Chemical and Biological Engineering. # Current address: University of Alberta, 315 Heritage Medical Research Centre, Edmonton, Alberta, Canada. O Current address: Tibotec Generall de Wittelaan, L-11, 2800 Mechelen, Belgium. 1 Abbreviations: BMCC, bacterial microcrystalline cellulose; CBM, carbohydrate-binding module; CBD, cellulose-binding domain; IPTG, isopropyl 1-thio--D-galactopyranoside; ITC, isothermal titration cal- orimetry: Ka, association constant; No, binding capacity; PASA, phosphoric acid swollen cellulose. 6240 Biochemistry 2001, 40, 6240-6247 10.1021/bi0101695 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/04/2001