Structural Properties of AMP-Activated Protein Kinase. Dimerization, Molecular Shape, and Changes upon Ligand Binding



Uwe Riek1,3, Roland Scholz1, Peter Konarev4,5, Arne Rufer6,Marianne Suter1, Alexis Nazabal2, Philippe Ringler7,Mohamed Chami7, Shirley A. Muller7, Dietbert Neumann1,Michael Forstner8,Michael Hennig6,Renato Zenobi2, Andreas Engel7, Dmitri Svergun4,5, Uwe Schlattner1,3, and Theo Wallimann1


  1. Institute of Cell Biology, Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
  2. Department of Analytical Chemistry, Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
  3. INSERM, U884, Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier, Grenoble, France
  4. EMBL, DESY, 22603 Hamburg, Germany
  5. Institute of Crystallography, Russian Academy of Sciences, 11733 Moscow, Russia
  6. F. Hoffmann-La Roche AG, Pharma Research Discovery Chemistry, 4070 Basel, Switzerland
  7. M.E. Muller Institute for Structural Biology, University of Basel, 4056 Basel, Switzerland
  8. Zurich Financial Services, 8002 Zurich, Switzerland


Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing γ1, (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (∼1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by ∼5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr172, thus positively affecting AMPK activity.

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AMPK Complex was chemically cross-linked using the CovalX K100 Stabilization Kit. 10 μL of AMPK complex at 2 μM was stabilized and then incubated for 1 hour on ice. 1 μL of the mixture was mixed with 1 μL of matrix (sinapic acid (10 mg/ml) in acetonitrile/deionized water 1:1 and 0.1% TFA) before 1 μL of this final mixture was spotted on a MALDI plate and allowed to dry. The samples were analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system. The data were background subtracted and smoothed using the CovalX Complex Tracker software.

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Categories : Publications, High-Mass MS