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J. Biol. Chem., Vol. 283, Issue 26, 18331-18343, June 27, 2008

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Structural Properties of AMP-activated Protein Kinase

DIMERIZATION, MOLECULAR SHAPE, AND CHANGES UPON LIGAND BINDING^*

Uwe Riek, Roland Scholz, Peter Konarev^¶||, Arne Rufer^**, Marianne Suter, Alexis Nazabal, Philippe Ringler, Mohamed Chami, Shirley A. Müller, Dietbert Neumann, Michael Forstner^¶¶, Michael Hennig^**, Renato Zenobi, Andreas Engel, Dmitri Svergun^¶||, Uwe Schlattner¹², and Theo Wallimann¹³

From the Institute of Cell Biology, and the Department of Analytical Chemistry, Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland, INSERM, U884, Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier, Grenoble, France, ^¶EMBL, DESY, 22603 Hamburg, Germany, and ^||Institute of Crystallography, Russian Academy of Sciences, 11733 Moscow, Russia, ^**F. Hoffmann-La Roche AG, Pharma Research Discovery Chemistry, 4070 Basel, Switzerland, the M. E. Müller Institute for Structural Biology, University of Basel, 4056 Basel, Switzerland, and the ^¶¶Zürich 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 ₁, (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.

Received for publication, October 9, 2007 , and in revised form, February 25, 2008.

^* This work was supported in part by European Union (EU) FP6 contract LSHM-CT-2004-005272 (EXGENESIS) and Swiss National Science Foundation (NSF) Grants 3100AO-102075 (to T. W. and U. S.), 3100+0-11437/1 (to T. W. and D. N.), and 501 221 (to A. E.); the French Agence Nationale de Recherche (ANR) "chaire d'excellence" (to U. S.); a graduate training fellowship from ETH Zurich (to U. S. and T. W.); a grant from the French Ministry of Education and Research (U. S.); EU Design Study SAXIER Contract 011934 (to D. S. and P. K.); and the Maurice E. Müller Foundation of Switzerland (to A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors are considered senior authors.

² To whom correspondence may be addressed: INSERM, U884, Laboratory of Fundamental and Applied Bioenergetics, University Joseph Fourier, BP 53, F-38041 Grenoble Cedex 9, France. E-mail: uwe.schlattner{at}ujf-grenoble.fr.

³ To whom correspondence may be addressed: Inst. of Cell Biology, ETH Zurich, Schafmattstrasse 18, 8093 Zurich, Switzerland. E-mail: theo.wallimann{at}cell.biol.ethz.ch.

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