AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the beta-subunit, for which there are two isoforms (beta(1) and beta(2)). Muscle-specific beta(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous beta(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of beta(2)-CBM is concurrent with an increase in flexibility in beta(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to beta(1)-CBM, unbound beta(2)-CBM showed microsecond-to-millisecond motion at the base of a beta-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this beta-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from beta(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the beta(2)-CBM mutant. Insertion of a threonine into the background of beta(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.