Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an alpha beta gamma heterotrimer that is important in regulating energy metabolism in all eukaryotes. The beta-subunit exists in two isoforms (beta 1 and beta 2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (beta 1- and beta 2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. beta 2-CBMbinds linear carbohydrates with 4-fold greater affinity than beta 1-CBM and binds single alpha 1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of beta 2-CBM in complex with the single alpha 1,6-branched carbohydrate glucosyl-beta-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the alpha 1,6 branch bound. We therefore solved the X-ray crystal structures of beta 1- and beta 2-CBM, in complex with gBCD, to 1.7 and 2.0 angstrom (1 angstrom = 0.1 nm) respectively. The additional threonine (Thr(101)) of beta 2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the alpha 1,6 branch. Hydrogen bonds are formed between the alpha 1,6 branch and the backbone of Trp(99) and Lys(102) side chain of beta 2-CBM. In contrast, the alpha 1,6 branch could not be observed in the beta 1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to beta 1- and beta 2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific beta 2-isoform may have greater affinity for partially degraded glycogen.