Structural, Biochemical, and Functional Studies on the Regulation of the S. Cerevisiae AMPK Homolog SNF1

Abstract

The 5’AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis. AMPK is activated by a high AMP:ATP ratio, and functions as a metabolic thermostat. By sensing when energy is low, AMPK upregulates energy-producing pathways (e.g., glycolysis, glucose transport, fatty acid oxidation, food intake) while downregulating energy-consuming pathways (e.g., gluconeogenesis, fatty acid synthesis). Due to its central role in controlling these processes, AMPK represents a key drug target for both diabetes and obesity. In S. cerevisiae, the AMPK homolog Sucrose Non-Fermenting 1 (SNF1) controls many of the same pathways as AMPK and, like AMPK, is a heterotrimeric protein comprised of a catalytic alpha subunit and regulatory beta and gamma subunits. We present here structures of the heterotrimer core of SNF1 and the catalytic protein kinase domain/auto-inhibitory domain (KD-AID) of the alpha subunit. Our studies elucidate important differences between SNF1 and higher eukaryotic AMPKs, especially with regards to AMP activation. In addition, we provide the first structural insight into the Regulatory Sequence (RS) of the alpha subunit, a region that interacts with the gamma subunit of SNF1. GST pulldown experiments demonstrate strong, direct interactions between the RS and the heterotrimer core. These interactions can be greatly reduced in vitro by the introduction of single-site mutations, although no effect is observed in vivo. We also probed the role of an AID N-terminal to the RS through crystallographic studies of a KD-AID protein. Interestingly, the AID in this structure is disordered, but the KD reveals a novel DFG-out conformation blocking ATP binding to the active site. Together, these data indicate that the RS is constitutively bound to the SNF1 gamma subunit, and the AID may be required to regulate SNF1 activity.