Structure of mammalian AMPK and its regulation by ADP

Bing Xiao et al:nature,472,14 Apr 2011

AMP-activated protein kinase (AMPK) was first discovered as an activity that inhibited preparations of acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) and was induced by AMP.

AMPK induces a cascade of events within cells in response to the ever changing energy charge of the cell.

a central control point in maintaining energy homeostasis.

recent evidence has shown that AMPK activity can also be regulated by physiological stimuli, independent of the energy charge of the cell, including hormones and nutrients.

5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate-activated protein kinase is an enzyme that plays a role in cellular energy homeostasis.

It consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans.

expressed in a number of tissues, including the liver, brain, and skeletal muscle.

Net effect of AMPK activation a. stimulation of hepatic fatty acid oxidation and

ketogenesisb. inhibition of cholesterol synthesis, lipogenesis and

triglyceride synthesisc. inhibition of adipocyte lipolysis and lipogenesisd. stimulation of skeletal muscle fatty acid oxidation and

muscle glucose uptakee. and modulation of insulin secretion by pancreatic beta-

cells.

The mammalian AMPK is a trimeric enzyme composed of a catalytic α subunit and the non-catalytic β and γ subunits.

There are two genes encoding isoforms of both the α and β subunits-α1, α2, β1 and β2and three genes encoding isoforms of the γ subunit-γ1-γ3

there are multiple genes encoding each of the subunits of AMPK

it is possible that 12 different isoforms of the hetertrimeric enzyme can be formed.

The α2 isoform is the subunit found within skeletal and cardiac muscle,

pancreatic islet β-cells the α1 isoform predominates which is also the case for white adipose tissue.

γ3 subunit found almost exclusively in glycolytic skeletal muscle.

N-terminal half of the α subunits contains a typical ser/thr kinase catalytic domain.

Interaction with the β and γ subunits occurs via the C-terminal half of the α subunits.

yeast AMPK β subunits are lipid modified with myristic acid.

Myristoylation may account for the membrane association of mammalian AMPK.

The core of the β subunits have a glycogen-binding domain (GBD).

This domain is closely related to the isoamylase N domain subfamily and weakly related to domains in the glycogen-targeting phosphatase subunits and several starch-binding proteins.

The close proximity of AMPK to cellular glycogen stores allows it to rapidly effect changes in glycogen metabolism in response to changes in metabolic demands.

The γ subunits of AMPK have been shown to contain four tandem repeats that form nucleotide binding sites with similarity to cystathionine β-synthase which are therefore referred to as CBS domains.

direct AMP-binding studies have shown that AMP is bound to the γ subunits by a pair of CBS domains.

The CBS domains also bind ATP and the binding of AMP and ATP to AMPK occurs in a mutually exclusive manner.

Binding of ATP keeps the activity of AMPK low and when AMP levels rise the exchange of AMP for ATP results in a 5-fold increase in kinase activity

clinical significance is the observation that mutations in the CBS domains of the γ2 subunit (gene symbol PRKAG2) are associated with Wolff-Parkinson-White syndrome and familial hypertrophic cardiomyopathy.

An additional inherited disorder associated with mutations in the PRKAG2 gene is a severe cardiac condition called lethal congenital glycogen storage disease of the heart. This disorder is caused by a single amino acid substitution of glutamate for arginine at position 531 (R531Q).

Function

AMPK acts as a metabolic master switch regulating several intracellular systems including the cellular uptake of glucose, the β-oxidation of FA and the biogenesis of (GLUT4) and mitochondria. The energy-sensing capabilityof AMPK depends on fluctuations in the AMP:ATP ratio that take place during rest and exercise

Upon activation, AMPK increases cellular energy levels by inhibiting anabolic energy consuming pathway and stimulating energy producing, catabolic pathways

A recent research paper on mice at Johns Hopkins has shown that when the activity of brain AMPK was pharmacologically inhibited, the mice ate less and lost weight.

When AMPK activity was pharmacologically raised the mice ate more and gained weight

Research in Britain has shown that the appetite-stimulating hormone ghrelin also affects AMPK levels.

The antidiabetic drug metformin (Glucophage) acts by stimulating AMPK,leading to reduced glucose production in the liver and reduced insulin resistance in muscle

A number of recent studies suggest that the herbal medicine berberine, also activates AMPK and glucose transport in muscles

Introduction The heterotrimeric (AMPK) has 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 processes

AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer

AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation

Here they show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation

their studies show that active mammalian 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.

they determined the crystal structure of an active AMPK complex.

The structure 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 biochemical and structural data they develop a model for how the energy status of a cell regulates AMPK activity.

AMPK is regulated by a diverse range of hormones—for example, leptin,adiponectin, ciliary neurotrophic factor and ghrelin

it has a role in appetite glucose, lipid and protein metabolism, cell growth, and cell polarity

AMPK is a heterotrimeric complex comprising an α-catalytic subunit and two regulatory subunits (β and γ)

Activation of AMPK requires phosphorylation of Thr172, which lies in the activation segment of the N-terminal kinase domain of the α-subunit

Phosphorylation of Thr172 leads to a several-hundred-fold increase in activity

In mammals, calcium/ calmodulin-dependent protein kinase kinase-β (CaMKKβ) and LKB1 are the predominant kinases upstream of AMPK

Previous studies have shown that AMP protects against the dephosphorylation of Thr172 and we recently provided evidence that protection against dephosphorylation is the major physiological mechanism for activation of AMPK

In addition to activation by phosphorylation, AMP causes a 2–5-fold allosteric activation of AMPK depending on the nature of the isoforms present in the AMPK complex.

To address this they investigated the nucleotide-binding properties of the γ1 subunit of AMPK and determined the structure of the regulatory core of mammalian AMPK

Notably, their studies revealed that three of the four potential nucleotide-binding sites are occupied.

One of these sites contains a permanently bound AMP molecule (site 4, ) whereas AMP and Mg-ATP compete for binding at the other two sites (site 1 and site 3).

Unlike AMP, ADP has no significant allosteric effect on AMPK isolated from rat liver

Consistent with this, they also found that ADP does not activate recombinant AMPK under conditions where AMP produces a twofold activation(a)

However, their studies show that ADP provides protection of AMPK from dephosphorylation across a similar range of concentrations as AMP ( b).

they have also shown the same effect using AMPK purified from rat liver

a. AMP, but not ADP, allosterically activates AMPK.b. AMP and ADP protect AMPK from dephosphorylation. c. ATP does not protect against dephosphorylation. d. Mg-ATP competes with the protective effect of ADP on

dephosphorylation.

Although Mg-ATP does not protect against dephosphorylation ( c), it does compete with the protective effect of both AMP and ADP on dephosphorylation ( d). They have also shown that the protective effect of ADP is lost in a Wolff–Parkinson–White syndrome mutation they propose that AMP or ADP (AMP/ADP) binding shifts the equilibrium between dephosphorylation-sensitive and -insensitive states, and thus slows, but does not abolish, dephosphorylation of the enzyme by phosphatases.

Extending their earlier work looking at nucleotide binding to the regulatory fragment, they characterized binding of nucleotides to active full-length AMPK.

For these studies they used CaMKKβ to stoichiometrically phosphorylate Thr172 on the activation loop of recombinant full-length AMPK, they used the coumarin adducts of ATP and ADP as fluorescent reporters of nucleotide binding, and derived the binding parameters for the unlabelled nucleotides by competition experiments

they verified that the two species bind at the same sites by determining the crystal structures of the regulatory fragment in complex with coumarin-ADP and with ADP

The results show that the two exchangeable sites have markedly different affinities for nucleotides.

Binding at the tighter of the two sites is at least 30-fold stronger than at the weaker site.

Given that under physiological conditions most of the ATP is coordinated to Mg2+, and that the majority of AMP and ADP is not, they also measured nucleotide binding in the presence of this cation.

The data show that Mg-ATP binds up to tenfold weaker than ATP.

Thus, active AMPK binds AMP/ADP significantly more strongly than it does Mg-ATP at both exchangeable sites.

There are two lines of evidence that lead us to conclude that it is AMP/ADP binding at the weaker of the two exchangeable sites that accounts for the protection of the enzyme against dephosphorylation.

The second comes from our discovery that NADH binds to AMPK.NADH undergoes a significant change in fluorescence upon binding to AMPK.They used this property to establish that the cofactor binds to a single site on the enzyme, with a dissociation constant (Kd) of about 50μM. NADH binding is competed by AXPs binding to the stronger, but not the weaker, of the two exchangeable sites they repeated the ADP protection against dephosphorylation experiments using a range of NADH concentrations, we found no evidence for NADH competing with the protective effect of ADP on dephosphorylation, whereas NADH and ADP both compete with AMP for allosteric activation of the enzyme

This observation indicates that it is AMP/ADP binding at the weaker of the two exchangeable sites, the one that does not bind NADH, that is responsible for protection against dephosphorylation.

They also carried out co-crystallization of the regulatory fragment with one molar equivalent of ADP

The resulting electron density map showed full occupancy of ADP at site 1 and no detectable density at site 3, identifying site 3 as the weaker binding site.

They can therefore assign the allosteric effect to AMP binding at the tighter site 1, and protection against dephosphorylation is mediated by AMP/ADP binding at the weaker site 3.

Previous studies shown that AMP allosterically activates the enzyme whereas ADP does not.

However, phosphorylation remains central to AMPK regulation as the enzyme is inactive in the absence of Thr172 phosphorylation

Under optimal conditions, mammalian cells maintain ATP at a high level relative to ADP and AMP.

Typical concentrations of free adenine nucleotides in mammalian cells lie in the range of 3,000–8,000μM for ATP, 50–200μM for ADP and 0.5–5μM for AMP.

Because the free concentration of ADP is between 10- to 400-fold higher than AMP, and their binding constants are similar, ADP will be more successful at competing with Mg-ATP than AMP.

Therefore, ADP protects AMPK from dephosphorylation is likely to represent an important physiological mechanism for regulating the activity of the enzyme.

The activation loop mediates the interaction of the kinase domain with the regulatory fragment means that, in this conformation, Thr172 is protected from access by phosphatases.

This idea is strongly supported by site-directed mutagenesis experiments: mutation of β1 His233 (corresponding to His-235 in β2) at the interface with the kinase domain results in an enzyme that is activatable by phosphorylation but that has a significantly increased rate of dephosphorylation in phosphatase assays

Another component of the α-subunit–regulatory fragment interaction is a part of the segment of the α-chain that links the N-terminal kinase domain to the C-terminal regulatory fragment, involving residues between A373 and A382 that are largely conserved between α1 and α2 in vertebrates-called the α-hook structure

The α-hook interacts with the γ-subunit at the exchangeable binding site 3

The hook makes a lid over the nucleotide-binding site that accounts for a buried surface area of about 500Å

To test the role of the α-hook in mediating protection against dephosphorylation they generated a mutant in this region . The resulting enzyme was allosterically activated by AMP but was not subject to protection against dephosphorylation by AMP or ADP.

the mutation at His233 retain some protective effect of AMP/ADP

Given that this mutation would be expected to weaken the interaction between the kinase domain and the regulatory fragment, but not block it, it seems reasonable that AMP/ADP binding would still help to order the α-hook and thus facilitate the recruitment of the kinase domain.

Regulation of AMPK activation. AMPK is activated by phosphorylation of Thr 172 catalysed by

LKB1:STRAD:MO25 complex in response to increase in the AMP/ATP ratio and by CaMKKb in response to

elevated Ca2+ levels. Thr172 is dephosphorylated by PP2C protein phosphatase switching active AMPK to

the inactive form.

AMPK and the regulation of skeletal muscle metabolism: role of AMPK in the regulation of lipid and glucose metabolism in skeletal muscle. AMPK activity may be increased by an altered energy nucleotide or

by hormonal action. This activation of AMPK may result in an increase in glucose transport as well as an

increase in fatty acid oxidation.

SUMMARY

They demonstrated that the protective effect of AMP/ADP is mediated by its binding to the weaker of the two exchangeable site(site3). the α-hook region binds into this site in the presence of AMP and predict that the same situation would occur with ADP. also suggest that binding of the α-hook acts to restrict the flexibility of the preceding α-linker region (residues 300–370) and promotes the interaction of the kinase domain with the regulatory fragment seen in our crystal structure.

This interaction, which mostly involves contacts between the activation loop and the C-terminal domain of β, would therefore act to protect Thr172 against dephosphorylation.

there is a dynamic equilibrium between the α-hook-bound and α-hook-unbound species.

AMP/ADP binding favours the α-hook-bound species but Mg-ATP binding drives formation of the α-hook-unbound species, then the competitive binding of AMP/ADP versus Mg-ATP would control the extent to which the enzyme was protected from dephosphorylation and inactivation.

Methods AMPK complexes were expressed in E.coli BL21 (DE3) cells,

purified by affinity chromatography using nickel-Sepharose and phosphorylated by incubation with CaMKKβ.

AMPK activity was determined using 0.2 mM SAMS peptide9, 0.2 mMATP and 5mM MgCl2.

Dephosphorylation of AMPK by recombinant PP2C-α was monitored either by measuring AMPK activity using the SAMS peptide assay or by western blotting of phospho-T172.

Western blot signals for phospho-T172 and total AMPK α-subunit (determined using sheep anti-α1 or anti-α2 antibodies) were quantified using the Li-Cor Odyssey infrared imaging system.

Uncorrected fluorescence spectra of the nucleotides and NADH and their complexes were recorded at 20 °C using a Jasco FP-6300 fluorimeter.

Dissociation constants for AMP, ADP and ATP were determined using competition assays.

The engineered crystallization construct was expressed as a His-tag fusion protein in E. coli.

Purified protein was phosphorylated using CAMKKβ before mixing with AMP and staurosporine.

Crystals were grown by the hanging-drop method using isopropanol and MPD as precipitant.

Diffraction data were collected on the Diamond Light Source, .

The structure was solved by molecular replacement using Amore29 and standard refinement was carried out with Refmac530 with manual model building with COOT