Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding. - Archive ouverte HAL Access content directly
Journal Articles Journal of Biological Chemistry Year : 2008

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.

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Abstract

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 gamma(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 approximately 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 Thr(172), thus positively affecting AMPK activity.

Dates and versions

inserm-00390926 , version 1 (03-06-2009)

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Cite

Uwe Riek, Roland Scholz, Peter Konarev, Arne Rufer, Marianne Suter, et al.. Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.. Journal of Biological Chemistry, 2008, 283 (26), pp.18331-43. ⟨10.1074/jbc.M708379200⟩. ⟨inserm-00390926⟩

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