Structure of mammalian AMPK and its regulation by ADP

Abstract

The heterotrimeric AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy producing pathways and inhibits energy consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer 2,3,4,5,6. AMPK is converted from an inactive to catalytically competent form by phosphorylation of the activation loop within the kinase domain7; AMP binding to the γ regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation as well as causing allosteric activation9. We show here that ADP binding to just one of the two exchangeable AXP binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active AMPK displays significantly tighter binding to ADP than to Mg.ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg.ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. It shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity (Supplementary Fig. 1).

AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD + elevation

“…Adenosine 5' monophosphate‐activated protein kinase (AMPK) serves as a cellular energy sensor, which is composed of a catalytic α subunit and regulatory β and γ subunits (Xiao et al ., 2011). The role of AMPK in preventing aging/senescence has been suggested in many studies (Apfeld et al ., 2003; Stenesen et al ., 2013; Ido et al ., 2015).…”

Adenosine Monophosphate-Activated Protein Kinase: A Central Regulator of Metabolism with Roles in Diabetes, Cancer, and Viral Infection

“…Although each molecule of bound AXP interacts with side chains from more than one CBS repeat, a useful convention (Kemp et al 2007) is to number the sites according to the CBS repeat bearing the aspartate residue interacting with the ribose ring. Recent binding studies have helped to define the functions of the three sites (Xiao et al 2011). Site 1 binds all three AXPs with similar high affinities and, based on the concentrations of AMP that cause allosteric activation, binding at this site appears to be responsible for this effect, which is only observed with AMP Xiao et al 2011).…”

“…The "a hook" region of this linker interacts with AMP or ADP bound at site 3 ( Fig. 3; Xiao et al 2011). Phosphorylation of a serine that lies within a flexible loop within the a-carboxy-terminal domain (a-CTD; Ser485 in human a1) appears to inhibit phosphorylation at Thr172 (Horman et al 2006).…”

“…Recent binding studies have helped to define the functions of the three sites (Xiao et al 2011). Site 1 binds all three AXPs with similar high affinities and, based on the concentrations of AMP that cause allosteric activation, binding at this site appears to be responsible for this effect, which is only observed with AMP Xiao et al 2011). Site 2 is the empty site, whereas site 3 again binds all three AXPs with similar affinities, although their affinities are about 50-fold lower than at site 1.…”

Coordinated regulation of AMPK activity by multiple elements in the α-subunit

“…Our structure-based mutagenesis studies have demonstrated that two of the three nucleotide-binding sites, γ-site 3 and γ-site 4, are important for AMPK allosteric activation. Xiao et al [8] reported the structure of an active AMPK heterotrimer, comprising the kinase domain, AID and the regulatory α-linker of the α-subunit in addition to the core fragment, which revealed additional intersubunit interactions involving the activation loop of the kinase domain and a small segment within the α-linker. However, in the same study, it was concluded that mammalian AMPK does not contain an AID domain, inconsistent with our results [3].…”