Structural properties of AMP-activated protein kinase. Dimerization, molecular shape, and changes up
March 29th, 2008 | by admin |Structural properties of AMP-activated protein kinase. Dimerization, molecular shape, and changes upon ligand binding.
Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Recently, we developed a bacterial co-expression strategy for functionally intact full-length mammalian AMPK with a His-tag. Here we report on (i) the bacterial expression of untagged mammalian AMPK isoform combinations, all containing 1, (ii) a fully automated 4-dimensional purification protocol, and (iii) a detailed 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 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, showed a similar molecular shape of AMPK heterotrimers revealing elongated, flat particles with protrusions and indentations. 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. Based on our findings 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 T172, thus positively affecting AMPK activity.
Riek U, Scholz R, Konarev P, Rufer A, Suter M, Nazabal A, Ringler P, Chami M, Müller SA, Neumann D, Forstner M, Hennig M, Zenobi R, Engel A, Svergun D, Schlattner U, Wallimann T.
LBFA, Inserm U884, University Joseph Fourier, Grenoble cedex 9 38041.