Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.
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.
Riek
Uwe
U
Institute of Cell Biology, Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland.
Scholz
Roland
R
Konarev
Peter
P
Rufer
Arne
A
Suter
Marianne
M
Nazabal
Alexis
A
Ringler
Philippe
P
Chami
Mohamed
M
Müller
Shirley A
SA
Neumann
Dietbert
D
Forstner
Michael
M
Hennig
Michael
M
Zenobi
Renato
R
Engel
Andreas
A
Svergun
Dmitri
D
Schlattner
Uwe
U
Wallimann
Theo
T
D055372:AMP-Activated Protein Kinases; D000818:Animals; D019281:Dimerization; D006801:Humans; D008024:Ligands; D008027:Light; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D008968:Molecular Conformation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012568:Schizosaccharomyces / Q000201:enzymology
D008024:Ligands; D009097:Multienzyme Complexes; C526222:PRKAG1 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases