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

Jesse I. Mobbs, Ann Koay, Alex Di Paolo, Michael Bieri, Emma J Petrie, Michael A Gorman, Larissa Doughty, Michael W. Parker, David I Stapleton, Michael D W Griffin, Paul Raymond Gooley

Research output: Contribution to journalArticleResearchpeer-review

16 Citations (Scopus)

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-CBMbinds 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.

Original languageEnglish
Pages (from-to)245-257
Number of pages13
JournalBiochemical Journal
Volume468
Issue number2
DOIs
Publication statusPublished - Jun 2015
Externally publishedYes

Keywords

  • AMP-activated protein kinase (AMPK)
  • Carbohydratebinding module (CBM)
  • Glycogen
  • Isothermal titration calorimetry
  • Nuclear magnetic resonance spectroscopy
  • X-ray crystallography

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