AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimeric enzyme (alpha, beta, gamma subunits) essential in sensing and regulating metabolic processes. Two mammalian beta-subunit isoforms exist, each containing a central carbohydrate-binding module (CBM) which share 80% sequence identity. We have shown that the muscle-specific beta2-CBM isoform, either as an isolated domain or in the heterotrimer, binds glycogen mimetics up to ~20-fold more tightly than the ubiquitous beta1-CBM (1,2). Additionally, we observe that both beta1- and beta2-CBM bind optimally to single alpha,1-6 branched oligosaccharides, a conformation only observed during glycogen breakdown, suggesting ligand specificity.
The available NMR solution and X-ray crystal structures have not revealed any differences to account for these affinities, and indeed the ligand contact residues are strictly conserved. An apparent difference between the two isoforms is a threonine insertion in position 101 within a loop of beta2-CBM which is not directly involved in ligand binding. Using 15N-relaxation dispersion NMR experiments we find that beta2-CBM, but not beta1-CBM, shows significant μs motion (2). On binding ligand this motion is slowed, but becomes more widespread. We have hypothesized that beta2-CBM can adopt conformational states that are poorly accessed by beta1-CBM, thus accounting for the affinity differences. To test this hypothesis we are determining additional NMR and Xray structures of mutants and ligand complexes and characterizing the ligand on and off rates by SPR and substoichiometric 15N relaxation dispersion experiments. The results of these experiments will be presented.