The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.
AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase responsible for regulating metabolism in response to energy supply and demand. During metabolic stress the cellular AMP/ATP ratio increases leading to activation of AMPK, which in turn switches off energy-consuming anabolic pathways and switches on catabolic pathways to restore ATP levels. The AMPK αβγ heterotrimer comprises a catalytic α-subunit associated with β and γ regulatory subunits (Fig. 1a). Adenine nucleotides (ATP, ADP and AMP) bind interchangeably to the γ subunit where three of the four cystathionine β-synthase (CBS) tandem-repeat sequences provide nucleotide-binding sites1. Allosteric activation of AMPK by AMP appears to involve all three sites but site 3 may be the most important2,3. AMP binding initiates AMPK signalling by promoting phosphorylation of AMPK on α-Thr172 by upstream kinases LKB1 and Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). Once phosphorylated on α-Thr172 AMPK is further activated by AMP, and AMP sustains AMPK signalling by inhibiting dephosphorylation of α-pThr172 (ref. 4). AMP stimulation of αThr172 phosphorylation is dependent on N-terminal myristoylation of the β-subunit, since we demonstrated that AMP stimulation by both upstream kinases is lost following β-G2A mutation2. ATP on the other hand opposes allosteric activation by AMP as well as promoting the dephosphorylation of α-Thr172 (ref. 5). The α-regulatory subunit-interacting motif 2 (αRIM2; Fig. 1a)6 is required for sensing the adenine nucleotide bound state of the γ-subunit and transducing this signal to the α-catalytic domain, resulting in either stimulation (AMP) or inhibition (ATP) of AMPK activity1,6,7,8.
There has been keen interest in developing AMPK-activating drugs for potential therapeutic use in treating metabolic diseases including type 2 diabetes, obesity and cardiovascular disease. A number of small-molecule activators have been identified9 and two of these, A769662 (Abbott Laboratories) and 991 (Merck Sharp and Dohme Corporation and Metabasis Therapeutics), were shown in milestone structures to bind to a site formed between the small lobe of the α-subunit kinase domain and the β-subunit carbohydrate-binding module (CBM)8,10 termed the allosteric drug and metabolite-binding site (ADaM)11. Salicylate, the active metabolite of aspirin, is also thought to bind to this site12. ADaM site stabilization is enhanced by phosphorylation of the β-subunit residue Ser108 (refs 13, 14). We have shown that the need for β-pSer108 can be bypassed and that dephosphorylated AMPK (or dephosphorylation mimic α1-T172Aβ1-S108Aγ1 mutant) can be activated synergistically by A769662 and AMP whereas there is negligible activity with either activator alone13.
Another potent small-molecule allosteric activator of AMPK, 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (compound 2, C2, Metabasis Therapeutics), was identified by screening an AMP mimetic library15. It is thought that C2 activates both AMPK α1 and α2 isoforms in cell-free assays by binding to the γ-subunit. Although C2 is impermeable to cells, its corresponding di-iso-propyl phosphoester prodrug (compound 13, C13) preferentially stimulates AMPK α1 in mammalian cells16. AMP and C2 likely share a common AMPK-activating mechanism as their allosteric effects are not additive, and sensitivity to both is lost in the γ2-R531G AMP-insensitive mutant16. The purpose of our study was to identify the C2-binding site(s) on AMPK and investigate the relationship between activation of AMPK by A769662 and C2.
We have determined the crystal structure of the C2: AMPK complex, revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results raise the possibility that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.
Allosteric activation of AMPK
We found that C2 activates both AMPK γ1 and γ2 complexes (α1β1γ1 and α1β2γ1; α1β1γ2 and α1β2γ2) to a similar degree as AMP, whereas neither C2 nor AMP-activated γ3-containing complexes (α1β1γ3 and α1β2γ3) (Fig. 1b). Lack of AMP activation of γ3-containing complexes has also been reported recently17 but not in all studies18,19.
Since ATP concentration was previously shown to alter AMP dependence5, we investigated C2 activation of AMPK α1 at low (20 μM) and physiological (2 mM) ATP concentrations. Comparison of dose–response curves revealed that the half-maximal concentrations for C2 activation were similar at both low and high ATP (52.3±7.9 and 50.3±4.4 nM for 20 μM and 2 mM ATP, respectively; Fig. 1c,d), implying that C2 and ATP binding is non-competitive. In contrast, previous studies have shown that the concentration of AMP required for half-maximal activation increases with higher concentrations of ATP, which is consistent with AMP/ATP competition at the γ-subunit allosteric sites5. We also observed that C2 activation becomes strongly co-operative at high ATP, as shown by a shift in the Hill coefficient from 1.4±0.3 (20 μM ATP) to 2.3±0.4 (2 mM ATP) (Fig. 1c,d). In contrast, AMP does not exhibit co-operative binding at high ATP concentrations (Hill coefficient 1.0±0.2; Supplementary Fig. 1 and Supplementary Table 1). We are uncertain of the structural basis for the effect of ATP on C2 co-operative binding as well as the kinetic basis for ATP not influencing AMP activation in a co-operative manner. The γ-subunit resembles a classic multimeric-enzyme with its 4 CBS tandem repeats. One interpretation is that ATP binding to the γ-subunit influences C2 co-operativity but we have been unable to obtain crystals with both C2 and ATP bound to the γ-subunit thus far. In the event ATP cannot bind to γ in the presence of C2 we cannot formally eliminate the possibility that ATP binding at the kinase active site (α-subunit) rather than the γ-subunit may induce C2 co-operative binding.
Synergistic activation of AMPK
We examined whether C2, like AMP, can synergize with A769662 to activate dephosphorylated AMPK (Supplementary Fig. 2a). Furthermore, we explored whether this synergistic activation is also present in the AMPK α2 isoform. Modest allosteric activations were observed with λ-phosphatase pre-treated AMPK α1 or α2 complexes individually incubated with AMP or C2 or A769662 (Fig. 2a). We attribute the larger A769662 response using AMPK α1 to residual β1-Ser108 phosphorylation persisting after λ-phosphatase treatment (Supplementary Fig. 2a). Synergistic activation occurred when the λ-phosphatase-treated AMPK α1 (basal activity ≈0.6 nmol min−1 mg−1) was co-incubated with combinations of AMP/A769662 (≈671 nmol min−1 mg−1 or≈1100-fold) and C2/A769662 (≈636 nmol min−1 mg−1 or ≈1040-fold), but not with CaMKK2-treated AMPK α1 (Fig. 2a,b; Table 1). It should be noted the maximum activity observed in C2-stimulated, CaMKK2-treated AMPK α1 is ≈threefold higher than the maximum activity observed with the λ-phosphatase-treated AMPK α1 co-incubated with C2/A769662. Moreover, the synergistic activation was specific to AMPK α1, as the λ-phosphatase-treated AMPK α2 (basal activity ≈1.2 nmol min−1 mg−1) co-incubated with AMP/A769662 or C2/A769662 was relatively insensitive to dual ligand activation (AMP/A769662 activation ≈7.2 nmol min−1 mg−1 or ≈6.1-fold; C2/A769662 activation ≈1.3 nmol min−1 mg−1 or ≈1.2-fold; Fig. 2a; Table 1). Similarly to CaMKK2-treated AMPK α1, the CaMKK2-treated AMPK α2 did not exhibit dual synergistic activation by both AMP/A769662 and C2/A769662 (Fig. 2b).
Novel C2-binding site in the γ-subunit
To gain further insight into the mechanism of C2-mediated AMPK activation we solved the X-ray crystal structure of full-length α2β1γ1 isoform co-crystallized with C2 and AMP to 2.99 Å resolution (Table 2). The structure revealed two molecules of C2, bound within the solvent-accessible core of the γ-subunit. The phosphate groups of both C2 molecules overlap the phosphate binding sites of AMP in sites 1 and 4, while the 5-(5-hydroxyl-isoxazol-3-yl)-furan moieties occupy novel binding sites independent from the AMP-binding sites (Fig. 3a–c; Supplementary Fig. 3a). The asymmetric unit contained two AMPK heterotrimers. One heterotrimer contained AMP bound at each of CBS sites 3 and 4 (Fig. 3a, white) whereas the other heterotrimer was devoid of AMP and fortuitously contained two molecules of C2 instead (Fig. 3a–c). The two distinct liganded complexes allowed us to comprehensively probe the differences between AMP and C2 binding to AMPK.
Our AMP-bound heterotrimer closely resembles the previously published AMP-bound α2β1γ1 structure (PDB 4CFE)8. The two molecules superpose with a r.m.s. deviation of 0.55 Å over 884 Cα atoms, with key AMP-interacting residues having the same conformation. In contrast, the C2-bound molecule has an r.m.s. deviation of 0.75 Å (over 864 Cα atoms) when superimposed with 4CFE8. The largest structural difference between the C2 and AMP-bound heterotrimer is in the β-CBM and the N-lobe of the kinase domain, where a shift of up to 4.0 Å occurs (Fig. 3a). This shift reflects the formation of a canonical kinase salt bridge, formed between conserved residues α-Lys45 and α-Glu64, in the C2-bound heterotrimer. This important interaction is regarded as an active kinase conformation signature20 and is conspicuously absent in previous α2β1γ1 structures8. The C2-bound γ1-subunit conformation closely mimics that of the AMP-bound complex. Despite the considerable chemical differences and different binding orientations between C2 and AMP the side chains of the γ1-subunit residues are remarkably similar in conformation, with the notable exceptions being γ-Arg70 and γ-His298 (Supplementary Fig. 4a,b). A region that does distinguish between the two heterotrimers is the nucleotide sensing α-subunit RIM2 motif1,6,8. Strong electron density was observed for the α-RIM2 in the AMP-bound heterotrimer, but only very weak electron density was observed for α-RIM2 in the corresponding C2-bound heterotrimer and could therefore not be included in this model (Supplementary Fig. 3b). This is a C2 specific effect as the previously published AMPK structure 4CFE (crystallized in the same space group (P21) and solved to an equivalent resolution of 3.02 Å) revealed strong electron density for both AMP-bound heterotrimers in the asymmetric unit8. Importantly, these data are consistent with the α2-subunit RIM2 being incompletely engaged with the γ-subunit when C2 is bound.
The two C2 molecules occupy the interface between the CBS-binding sites 1, 3 and 4, with the phosphate group of C2 occupying a similar position to that of AMP-bound phosphate group in sites 1 and 4 (Fig. 3b). For this reason we have named specific C2-binding sites γ-pSite-1 and γ-pSite-4, referring to the equivalent AMP phosphate group position (Fig. 3b,c).
In both sites the C2 molecule makes six protein-mediated hydrogen bond contacts, while C2 in γ-pSite-4 makes an additional π-stacking interaction with γ-His298 (Fig. 3c). All of the residues that contact C2 are shared with AMP, yet the binding constant for C2 is >100-fold higher than that for AMP15,16. The basis of C2’s increased affinity is not obvious to us.
Mutational analysis of the C2-binding site
To validate the C2-binding sites, we expressed three AMPK γ1 mutants (T89E, H151E and H298E) in COS7 mammalian cells and measured their sensitivity to C2 or AMP. We initially found that all the γ1 mutants displayed increased but variable Thr172 phosphorylation and basal activities compared with the wild-type (WT) γ1-containing AMPK complexes. We therefore co-expressed the mutants with the phospho-mimetic AMPK α1(T172D), which is more amenable to studying the effect of these γ mutations on allosteric activation (Supplementary Fig. 2b)5,21. C2 and AMP activated the AMPK α1(T172D) β1γ1 enzyme to a similar extent (≈ 8- and 10-fold, respectively; Fig. 4a). The γ-T89E and γ-H298E mutants had increased basal activities (9.5±0.2 and 11.4±0.2 nmol min−1 mg−1 lysate for γ-T89E and γ-H298E, respectively) in the absence of C2 and AMP compared with γ1 WT (1.4±0.1 nmol min−1 mg−1 lysate), and were insensitive to further activation by either C2 or AMP (Fig. 4a). It is possible that substituting for a negatively charged glutamate residue at the T89 and H298 positions mimics the effect of the phosphate groups of AMP or C2, resulting in constitutive activation. This would also explain the increased Thr172 phosphorylation observed with these mutants. The γH151E mutation completely abolished AMPK activation by C2 (Fig. 4a,c), while only partially reducing AMP-dependence and sensitivity compared to the wild-type control (Fig. 4a,b).
C2-induced conformational rearrangements of the γ-subunit
A superposition of the two AMPK heterotrimers reveals two major side-chain conformational changes between AMP and C2 binding. Firstly, the imidazole ring of the aforementioned γ-His298 has flipped 180° and rotated at the Cβ>90° (∼ 6.0 Å) to form a π-stacking interaction with C2 in γ-pSite-4 while simultaneously blocking site 3, mimicking ATP-bound AMPK (PDB 4EAK)3 (Supplementary Fig. 4a). The conformational difference of the γ-His298 side chain is critical for AMP or C2 binding. Secondly, the position of C2 at γ-pSite-1 causes the side-chain guanidine group of γ-Arg70 to shift approximately 4 Å away from the AMP-bound conformation. In this new conformation, γ-Arg70 would clash with critical AMP-sensing residue α-Glu368 from the αRIM2 (Supplementary Fig. 4b). The clash may cause α-Glu368 to lose hydrogen bonds to site 3 AMP-interacting residues γ-Arg70 and γ-Lys170, that tether α-Glu368 to the γ-subunit1,6,8. In turn, this causes the αRIM2 disengagement. The loss of RIM2 association with the γ-subunit leads to the loss of protection from α-Thr172 dephosphorylation and a concomitant reduction in Vmax with C2 as shown by Hunter et al.16. In support of this proposal, previous studies have shown that α-Glu368 mutation results in a comparable reduction in Vmax with AMP stimulation and α-Thr172 dephosphorylation protection6,7,8.
AMPK isoform specificity of C2 allosteric activation
Secondary structure elements of αRIM2 are crucial for the isoform specificity of C2. Structural comparison of α1 and α2 isoform αRIM2/γ-subunit interactions reveal the α-helical α1RIM2 (PDB 4CFH)1 forms a strong interaction (three hydrogen bonds and six salt-bridges)22 with the γ-subunit in a groove between γ-helix α10 and γ-helix α4, formed upon AMP or C2 binding (Supplementary Fig. 3b,c). In contrast, α2 isoforms have a loop motif (PDB 4ZHX and 4CFE)8 that forms a single hydrogen bond and four salt-bridges22 with the γ-subunit (Supplementary Fig. 3b,c). We propose the weaker interacting α2 isoform αRIM2 is incompletely engaged upon C2 binding relative to the α1 isoform, resulting in the α-isoform specificity for allosteric and synergistic activation and protection from α-Thr172 dephosphorylation. We solved the X-ray crystal structure of a heterotrimeric α2/α1 RIM chimaera, α2(1-347)/α1(349-401)/α2(397-end) β1γ1 (which has previously been shown to completely restore maximal allosteric activation and C2-mediated protection of α-Thr172 dephosphorylation16), co-crystallized with C2 to 2.99 Å (pdb: 5EZV; Table 2). Unlike the aforementioned crystal structure (4ZHX), α2/α1 RIM swap chimaera was crystallized in the absence of AMP, revealing two C2-bound AMPK heterotrimers in the asymmetric unit. Both of the C2-bound heterotrimers (5EZV) closely resembled the previous C2-bound heterotrimer (molecule 1 4ZHX), with an r.m.s deviation of 0.520 over 869 Cα atoms (molecule 1 5EZV). The C2 molecules occupy the same binding space and γ-subunit residue interactions as the AMP/C2-bound structures (4ZHX). Importantly the structure reveals density for seven α1 RIM 2 residues (Fig. 5), including the main-chain of the nucleotide sensing α-E364 (α2 E368), further supporting our proposal that the α1 RIM2 interacts more strongly with the γ-subunit. Interestingly there was weak density for the carboxyl group of the E364 side-chain, possibly indicating a transient interaction with the γ-subunit residue R70.
In this study we showed that two topographically disparate drug sites on AMPK, one located at the classic α-kinase domain/β-CBM interface (ADaM site) and the other located within the solvent-accessible core of the γ-subunit (γ-pSite-1/γ-pSite-4), can be exploited to achieve synergistic activation of unphosphorylated α1-AMPK independent of AMP and upstream phosphorylation events. This provides a major advance to previous demonstrations of AMPK synergistic activation that have been dependent on AMP. Importantly, identification of C2-binding sites on the γ-subunit represents an entirely unexplored dimension for future rational drug design.
Antibodies for pan AMPK α (#2793), FLAG (#2368) and HA (#2367); phosphospecific antibodies for α-pThr172 (#2535) and β-pSer108 (#4181), and λ-phosphatase (#P0753) were from Cell Signaling Technology. β1 antibody (#ab58175) and A769662 (#ab120335) were from Abcam. Anti-rabbit and anti-mouse IgG secondary antibodies fluorescently labelled with IR680 or IR800 dye, respectively, were from LI-COR Biosciences. Glutathione Sepharose 4B was from GE Life Sciences. FLAG peptide (DYKDDDK) was provided by GL Biochem (Shanghai), purified by reversed-phase chromatography and stored as lyophilized powder. All other reagents were purchased from Sigma.
Constructs for crystallization
All mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. Escherichia coli-expressed AMPK preparations were mass verified by time-of-flight (TOF) mass spectrometry. Heterotrimeric human AMPK His6-α2β1γ1 was expressed in E. coli strain Rosetta (DE3) using the pETDuetTM-1 expression system (Novagen) as previously described13. Briefly, cDNAs for α2 and γ1were sequentially inserted into pETDuetTM-1 multiple cloning sites (MCS) 1 (BamH1/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2. cDNA for β1 was inserted into pRSFDuetTM-1 MCS1 (NcoI/ BamHI). Heterotrimeric human AMPK α2/α1 RIM chimaera (α2(1-347)/α1(349-401)/α2(397-end) β1γ1)16 was expressed in E. coli strain Rosetta (DE3) using the pETDuetTM-1 and pCOLADuetTM (Novagen). cDNAs for α2/1 chimaera and γ1were sequentially inserted into pETDUETTM-1 multiple cloning sites (MCS) 1 (BamHI/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2/1 chimaera. cDNA for β1 was inserted into pCOLADuetTM-1 MCS1 (NcoI/ BamHI).
Protein expression and purification for crystallization
Expression cultures were grown in Luria-Bertani broth and induced at 16 °C with 0.25 mM isopropyl β-D-1-thiogalactopyranoside, before overnight incubation. Cells were lysed using a precooled EmulsiFlex-C5 homogenizer (Avestin) and AMPK purified using Nickel Sepharose and size exclusion chromatography in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM Tris(2-carboxyethyl)phosphine buffer. AMPK was phosphorylated by incubation with CaMKK2 (expressed in Sf21 cells as a C-terminal FLAG fusion and purified on FLAG monoclonal antibody-coupled affinity resin2) in the presence of 2.5 mM MgCl2, 0.5 mM ATP and 0.5 mM AMP (1 h, 22 °C). Protein was re-purified using size exclusion chromatography, concentrated to ∼10 mg ml−1 and flash frozen in liquid nitrogen before storage at −80 °C (storage buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM Tris(2-carboxyethyl)phosphine).
Full-length myristoylated and phosphorylated α2β1γ1 (4 mg ml−1) was mixed with a twofold molar excess of C2, AMP and S108tide (synthetic peptide: β1(102-117) R115-117, NH2-KLPLTRSHNNFVARRR-COOH) and a 1:1 molar ratio of Staurosporine and A769662, while full-length phosphorylated RIM swap chimaera (α2 1-347/α1 349-401/α2 397-end β1γ1)16 (4 mg ml−1) was mixed with a sixfold molar excess of C2, twofold molar excess of S108tide and a 1:1 molar ratio of Staurosporine and A769662. Crystals from both protein preparations were grown following the previously described method8. Briefly, protein was mixed equally at room temperature with a reservoir solution containing 8% PEG 3350, 0.1 M MgCl2, 1.0% glucose, 0.001% cocamidopropyl betaine and 0.1 M imidazole (pH 6.2). Protein crystals were incubated with reservoir solution containing an additional 5% glycerol, 5% PEG 400, 5% MPD, 5% sucrose, 5% sorbitol and 5% ethylene glycol for∼2 min before flash-cooling in liquid nitrogen. Data were collected at the Australian Synchrotron MX2 beamline, Melbourne, Australia. Data were processed and integrated using XDS23 and scaled using AIMLESS from the CCP4 suite24. The structure was solved by molecular replacement using Phaser from the CCP4 suite25 and 4CFE8 as the search model. Iterative rounds of model building and refinement were performed using Coot26 and Buster (https://www.globalphasing.com/buster/)27 respectively. C2 molecular coordinates and restraints were generated using the PRODRG web-server28. Omit maps were calculated using Sfcheck in the CCP4 suite29. Structure validation and protein–C2 interactions was performed using Molprobity30, figures were created using Pymol (https://www.pymol.org/). Data collection and refinement statistics are listed in Table 2.
Protein expression and purification for AMPK enzyme assays
All mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. Heterotrimeric AMPK was expressed in COS7 mammalian cells by transient transfection, using FuGENE HD (Promega) transfection reagent according to manufacturer’s protocol. Transfected constructs included N-terminal GST-α1 and α2 fusions2 (pDEST27 expression vector), C-terminal FLAG- α1 and α2 fusions (cloned into pcDNA3.1 expression vector using XhoI/EcoRI (α1) or XhoI/HindIII (α2) restriction sites, C-terminal myc-β1 and β2 fusions31 (pcDNA3.1 expression vector) and N-terminal HA-γ1, γ2 and γ3 fusions32 (pMT2 expression vector). To assess C2 γ isoform selectivity and C2 kinetics (Fig. 1): GST-α1β1/2γ1/2/3 AMPK complexes were isolated from transfected lysates by immobilization on Glutathione Sepharose 4B (GE Life Sciences) prior to SAMS activity assay2. To assess C2/A769662 synergy (Fig. 2): GST-α1β1γ1 and GST-α2β1γ1 AMPK complexes were immobilized on Glutathione Sepharose 4B and incubated with either λ-phosphatase (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 10% glycerol, 0.01% Tween-20 (buffer A) supplemented with 2 mM MnCl2) or CaMKK2 (buffer A supplemented with 2 mM MgCl2, 200 μM ATP) (1 h, 22 °C) prior to extensive washes with buffer A and elution in buffer A supplemented with 20 mM glutathione. The levels of α-pThr172 and β-pSer108 of treated AMPK complexes were assessed by immunoblot as described previously32 (Supplementary Fig. 2a). Briefly, membranes were simultaneously probed overnight at 4 °C with a phosphospecific primary antibody for α-pThr172 (1:2,000 dilution in phosphate buffered saline with 0.1% Tween-20 (v/v) (PBS-T)) or β-pSer108 (1:2,000 dilution in PBS-T) labelled with the fluorescent dye IR680 (LI-COR Biosciences) and probed for 1 h at room temp with pan α primary antibodies (1:1,000 dilution in PBS-T) or β1 primary antibodies (1:1,000 dilution in PBS-T) fluorescently labelled with IR800. Immunoreactive bands were visualized on an Odyssey membrane imaging system (LI-COR Biosciences) and densitometry analyses performed using integrated software. To evaluate the impact of γ-mutation on C2/AMP activity (Fig. 4): FLAG-α1(T172D)β1γ1 (wild-type and indicated mutants) AMPK complexes were immobilized on anti-FLAG M2 affinity Agarose gel (Supplementary Fig. 2b) before SAMS activity assay. The expression levels of the γ-mutants were assessed by immunoblot. Membrane was probed overnight at 4 °C with a FLAG primary antibody (1:2,000 dilution in PBS-T) labelled with the fluorescent dye IR680 (LI-COR Biosciences) and probed for 1 h at room temp with HA primary antibodies (1:1,000 dilution in PBS-T) fluorescently labelled with IR800 and analysed as above.
AMPK activity assay
AMPK activity assay was conducted as described previously13. Briefly, AMPK complexes were purified as above and washed three times with a 40:1 v/v ratio of assay buffer/resin (assay buffer: 50 mM Hepes pH 7.4, 1 mM DTT and 0.02% Brij-35). Reaction mixtures (25 μl) containing 100 μM SAMS synthetic peptide (sequence: NH2-HMRSAMSGLHLVKRR-COOH), 5 mM MgCl2, 200 μM [γ-32P]-ATP were incubated AMPK for 10 min at 30 °C in the absence or presence of various C2 concentrations. AMP (100 μM) and/or A769662 (20 μM) were included as appropriate positive controls. After 10 min, phosphotransferase activity was quenched by spotting onto P81 ion-exchange chromatography paper (Whatman, GE Healthcare, Parramatta, NSW) followed by repeated washes in 1% H3PO4 (ref. 33). The level of 32P-transfer to the SAMS peptide was determined by liquid scintillation counting (Perkin Elmer, Melbourne, VIC). Statistical tests were conducted using analysis of variance (ANOVA). The values for EC50 and fold stimulation were calculated based on the equation: Activity=Basal+(((Fold stimulation x Basal)−Basal × [C2])/((EC50)+[C2])). Curve fitting was performed using KaleidaGraph software.
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This research was partly undertaken on the MX2 beamline at the Australian Synchrotron, Victoria, Australia and we thank the beamline staff for their assistance. This work was supported by grants and fellowships from the Australian Research Council (ARC) and the National Health and Medical Research Council (NHMRC). Supported in part by the Victorian Government’s Operational Infrastructure
The authors declare no competing financial interests.
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