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 Schlattner,Theo Wallimann

doi:10.1074/jbc.m708379200

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.

Highlights

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms
In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by ϳ5% in small angle x-ray scattering (SAXS), which indicates a conformational switch in AMPK induced by ligand binding
AMPK eluted from the final calibrated gel filtration column at an elution volume corresponding to ϳ240 kDa

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Expression of Proteins in Bacteria—Tricistronic AMPK expression plasmids were constructed as described earlier [45], but encoding non-tagged versions of the four different mammalian AMPK isoform combinations ␣1␤1␥1, ␣2␤1␥1, ␣1␤2␥1, and ␣2␤2␥1 (GenBankTM accession numbers U40819, Z29486, X95577, AJ224538, and X95578). (Iexp experimental data as function of momentum transfers) Prior to this analysis, an appropriate constant was subtracted from each data point to force the s-4 decay of the intensity at higher angles following Porod law for homogeneous particles. An appropriate constant was subtracted from each data point to force the s-4 decay of the intensity at higher angles following Porod law for homogeneous particles This “shape scattering” curve was further employed to generate low resolution ab initio models of monomeric AMPK heterotrimers using the program DAMMIN [59], which represents the protein by an assembly of densely packed beads. The results from 10 runs for each type were averaged using the program DAMAVER [60], which superimposed the models pairwise to generate the most typical model by retaining the common structural features

RESULTS

DISCUSSION

Samples c