title: Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.
abstract: AMP-activated protein kinase (AMPK) is an 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 4-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 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) 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 Trp99 and Lys102 side chain of β2-CBM. In contrast, the α1,6 branch could not be observed in the β1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to β1- and β2-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 β2-isoform may have greater affinity for partially degraded glycogen.
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Jesse I
authors.initials: JI
authors.lastname: Mobbs
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Ann
authors.initials: A
authors.lastname: Koay
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Alex
authors.initials: A
authors.lastname: Di Paolo
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Bieri
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Petrie
authors.affiliation: ║St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gorman
authors.affiliation: ║St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
authors.forename: Larissa
authors.initials: L
authors.lastname: Doughty
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: ¶The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010, Australia.
authors.forename: David I
authors.initials: DI
authors.lastname: Stapleton
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.affiliation: *Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006003:Glycogen / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D009844:Oligosaccharides; D021122:Protein Subunits; D006003:Glycogen; D055372:AMP-Activated Protein Kinases