Structure of Mammalian AMPK and its regulation by ADP

Download Report

Transcript Structure of Mammalian AMPK and its regulation by ADP

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 (HMGCoA 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 ketogenesis
b. inhibition of cholesterol synthesis, lipogenesis and triglyceride
synthesis
c. inhibition of adipocyte lipolysis and lipogenesis
d. stimulation of skeletal muscle fatty acid oxidation and muscle
glucose uptake
e. 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 glycogentargeting 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 5fold 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 nucleotidebinding 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.
b.
c.
d.
AMP, but not ADP, allosterically activates AMPK.
AMP and ADP protect AMPK from dephosphorylation.
ATP does not protect against dephosphorylation.
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 fulllength 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 400fold 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 MgATP 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 αsubunit (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
