Role of hydrogen bonding in the interaction between a xylan binding moduleand xylan

NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and t wo aromatic residues that interact with its target ligand, xylan. To invest igate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant fo rms of CBM2b-1. The data show that the two aromatic residues, Trp 259 and T rp 291, play a critical role in the binding, and similarly that mutants N26 4A and T316A have no affinity for the xylose polymer. Interestingly, mutati ons E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers , but do influence the thermodynamics driving the protein-carbohydrate inte ractions. These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the followi ng. (1) For proteins whose ligands are bound on an exposed surface, hydroge n bonding confers little specificity or affinity. It also displays little c ooperativity, Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings. (2) Loss of hydrogen bonding inter actions leads to a redistribution of the remaining bonding interactions suc h that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds. (3) Changes in entropy and en thalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization.