posters 5th Asia-Pacific NMR Symposium 2013

Carbohydrate binding modules of AMPK have complex isoform specificity. (#177)

Jesse Mobbs 1 , Michael Bieri 1 , Ann Koay 1 , Larissa Doughty 2 , Mike Gorman 2 , Michael Parker 1 2 , Alexandre Di Paolo 1 , Michael Griffin 1 , David Stapleton 3 , Paul Gooely 1
  1. University of Melbourne, Melbourne, VIC, Australia
  2. St. Vincent's Institute of Medical Research, Fitzroy, Vic, Australia
  3. Dept. of Physiology, University of Melbourne, Parkville, Vic, Australia

AMP activated protein kinase is a protein complex that plays an important role in energy metabolism in nearly all eukaryotes. AMPK is a heterotrimeric protein consisting of three different subunits, catalytic alpha (α1, α2), regulatory beta (β1, β2) and regulatory gamma (γ1, γ2, γ3). Following activation by phosphorylation AMPK can be further regulated by the AMP:ATP ratio, hormonal signals and localisation/regulation by glycogen. Once activated AMPK increases ATP producing pathways while at the same time inhibiting ATP consuming pathways. AMPK is considered a good target for treating metabolic disorders such as type-2 diabetes.

The β subunit contains a carbohydrate binding module (CBM) that binds to glycogen. Past research has shown that the CBM is important for localizing AMPK to glycogen and/or regulating AMPK activity via glycogen. One of our aims is to further elucidate how the CBM interacts with glycogen and why this is important for AMPK function. Our lab has found that the isolated β2-CBM always binds to oligosaccharides more tightly than the β1-CBM. The greatest difference in affinity was found for oligosaccharides with a single α1,6 branch . Relaxation dispersion experiments show μs motion for β2-CBM but not for β1-CBM and that Thr101 is important for these motions.

Isothermal titration calorimetry has shown that V134 is important for carbohydrate binding affinity and that the α1,6 branch is more important to β2-CBM than β1-CBM. X-ray crystallography is used to determine the contact positions of the branched oligosaccharide glucosyl-beta-cyclodextrin. Here we use CPMG relaxation dispersion experiments at substoichiometric concentrations and surface plasmon resonance to determine binding rates for the oligosaccharides to CBM.