Structural study of the yeast Snf1 kinase: A member of the Snf1/AMPK family

The Snf1/AMPK kinases are a conserved family of serine/threonine kinases found in all eukaryotes. These kinases are switched on in response to nutrient stress by tightly regulated mechanisms which result in an allosteric conformational change. The Snf1/AMPK kinases have been implicated in a variety of metabolic human disorders. In this study, the crystal structure of the kinase domain from yeast Snf1 was determined, revealing a bi-lobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that also forms in solution as demonstrated by equilibrium sedimentation and in yeast cells as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved αC helix and the burial of the activation segment and substrate-binding site within the dimer suggest that it represents an inactive form of the kinase. Taken together, these studies identify another layer of kinase regulation within the Snf1/AMPK family. The Snf1/AMPK kinases are present as a heterotrimeric complex in cells. After attempts to purify the full length Snf1 kinase were unsuccessful, various Snf1 complexes were purified by co-expression of individual subunits using multi-cistronic plasmids. Snf1 complexes were expressed in sufficiently large amounts and were used for crystallization studies. Crystallization conditions for the Gal83/Snf1/Snf4 complex were identified and preliminary crystals were obtained