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

AMPK-activated protein kinase (AMPK) is a αβγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The β-subunit exists in two isoforms (β1 and β2) and contains a carbohydrate binding module (CBM) that interacts with glycogen. The two CBM isoforms (β1- and β2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate binding affinity. β2-CBM binds linear carbohydrates with four-fold greater affinity than β1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of β2-CBM in complex with the single α1,6 branched carbohydrate glucosyl-β-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of β1- and β2-CBM, in complex with gBCD, to 1.7 Å and 2.0 Å respectively. The additional threonine (Thr-101) of β2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp-99 and Lys-102 side chain of β2-CBM. In contrast the α1,6 branch could not be observed in the β1-CBM ...

Source: BJ Signal - March 16, 2015 Category: Biochemistry Authors: J I Mobbs, A Koay, A Di Paolo, M Bieri, E J Petrie, M A Gorman, L Doughty, M W Parker, D Stapleton, M D W Griffin, P R Gooley Tags: BJ Biomolecules Source Type: research