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.
Skip Nav Destination
Article navigation
June 2015
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
May 22 2015
Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase
Jesse I. Mobbs;
Jesse I. Mobbs
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
Search for other works by this author on:
Ann Koay;
Ann Koay
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
Search for other works by this author on:
Alex Di Paolo;
Alex Di Paolo
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
‡Eurogentec Biologics Division, Rue Bois Saint-Jean, 14, 4102 Seraing, Belgium
Search for other works by this author on:
Michael Bieri;
Michael Bieri
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
§CSL Behring AG, Wankdorfstrasse 10, CH-3000 Bern 22, Switzerland
Search for other works by this author on:
Emma J. Petrie;
Emma J. Petrie
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
Search for other works by this author on:
Michael A. Gorman;
Michael A. Gorman
║St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Search for other works by this author on:
Larissa Doughty;
Larissa Doughty
║St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Search for other works by this author on:
Michael W. Parker;
Michael W. Parker
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
║St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Search for other works by this author on:
David I. Stapleton;
David I. Stapleton
¶The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010, Australia
Search for other works by this author on:
Michael D.W. Griffin;
Michael D.W. Griffin
1
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
1Correspondence may be addressed to either of these authors (email mgriffin@unimelb.edu.au or prg@unimelb.edu.au).
Search for other works by this author on:
Paul R. Gooley
Paul R. Gooley
1
*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia
†Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia
1Correspondence may be addressed to either of these authors (email mgriffin@unimelb.edu.au or prg@unimelb.edu.au).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
March 02 2015
Revision Received:
March 13 2015
Accepted:
March 16 2015
Accepted Manuscript online:
March 16 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2015 Biochemical Society
2015
Biochem J (2015) 468 (2): 245–257.
Article history
Received:
March 02 2015
Revision Received:
March 13 2015
Accepted:
March 16 2015
Accepted Manuscript online:
March 16 2015
Citation
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 R. Gooley; Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase. Biochem J 1 June 2015; 468 (2): 245–257. doi: https://doi.org/10.1042/BJ20150270
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.