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Structural Basis of AMPK Regulation by Small Molecule Activators


Structural Basis of AMPK Regulation by Small Molecule Activators

Bing Xiao et al. Nat Commun.


AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.


Figure 1
Figure 1. Regulation of AMPK in vitro and in HEK293 cells.
(a) Structures of A-769662 and 991 compounds, the rings of 991 are numbered. (b) 991 (red) allosterically activates recombinant α2β1γ1 with half maximal activation (A0.5) of 0.09±0.02 μM compared to 0.39±0.03 μM for A-769662 (black). Results are the mean±s.e.m. from at least three independent experiments. Inset, 991 protects against pT172 dephosphorylation of α1β1γ1 (grey) and α2β1γ1 (white) complexes. (c) Top, 991 (grey bars) activates endogenous AMPK in HEK293 cells at lower doses than A-769662 (white). HEK293 cells were treated with varying concentrations of A-769662 or 991, and endogenous AMPK was immunoprecipitated from cell lysates using a pan-β-specific antibody. AMPK activity was measured using the SAMS peptide assay and results are shown as the fold activation (±s.e.m.) relative to untreated cells from at least three independent experiments. Bottom, 991 and A-769662 increase phosphorylation of endogenous AMPK Thr-172 (pT172) and the AMPK target ACC (pACC) in HEK293 cells (Supplementary Fig. 17). Blots were generated using a capillary-based western blot automated system (Simon, ProteinSimple). (d) HEK293 cells were transfected with myc-α1, FLAG-γ1 and either wild-type GFP-β1, GFP-β1 harbouring a mutation at Ser-108 (S108A) or GFP-β1 lacking the CBM (ΔCBM). Cells were treated with varying concentrations of the 991 activator. Top, complexes were immunoprecipitated using the FLAG-tag antibody and AMPK activity measured using the SAMS peptide assay. Results are shown as the fold activity compared with cells not treated with 991 (±s.e.m. from at least three independent experiments). 991 did not activate AMPK lacking the CBM (ΔCBM, black) in HEK293 cells. Higher concentrations of 991 were required to activate the AMPK β1 Ser-108 to alanine mutant (S108A, grey) in HEK293 cells compared with wild-type (white). Bottom, expression of the GFP-β1 subunit was monitored by western blot analysis in cells treated with and without 10 μM 991. An untransfected sample is included as a control.
Figure 2
Figure 2. 991 activator binding to AMPK.
(a) BLI data (average of four traces) for the binding of 991 (1.5 μM) to α1β1γ1. A double exponential fit (blue) gave kobs=0.148 s−1. The dependence of kobs on the concentration of 991 (inset) gave a kon value of 0.103±0.008 μM−1s−1 (average of 4 traces). Analysis of the dissociation phase (see Supplementary Methods) gave koff=0.0062±0.0012, s−1, giving a Kd of 0.06±0.012 μM for the binding of 991 to α1β1γ1. (b) CD titration of 20 μM α1β1γ1 with 991 (0–32 μM). The spectrum of the protein (red) is very different from the spectrum of AMPK:991 complex (blue). Analysing the signal change at 306 nm as a function of 991 concentration (inset—1:1 binding model) gave a Kd of 0.078±0.026 μM.
Figure 3
Figure 3. Structure of full-length AMPK complex with activator.
(a) Bar diagram indicating the three subunits that make up the complex. (b) Cartoon representation of full-length α2β1γ1 in complex with the activator 991, the domains of the three subunits are coloured according to (a). The orientation of the figure is similar to our earlier paper, so that the kinase domain is ‘upside-down’ with respect to the classical kinase orientation. The activator, which binds at the interface of the kinase and CBM, is shown in stick representation with its carbon atoms coloured magenta. Omit density (Fo-Fc) covering the 991 compound is contoured at 2.5 sigma and coloured blue (see also Supplementary Fig. 3). (c) Detailed view of 991 binding in a pocket generated at the interface between the CBM and the kinase domain, and making interactions with a cluster of hydrophobic residues from each domain; Ile-46(Kinase), Phe-90(Kinase), Leu-18(Kinase) and Val-11(Kinase) and Val-81(CBM), Val-113(CBM), Ile-115(CBM) and one of the side-chain carbon atoms (CG) of Thr-106(CBM). These hydrophobic interactions mainly involve ring-1 and ring-2 of the activator (Fig. 1a). The hydrophobic residues from the kinase (yellow) and CBM (green) are shown as sticks with surfaces, while (d) shows the same view and details the polar interactions that contribute to activator binding. Ring-3, and its linkage to ring-2, are involved in a number of polar interactions. Salt-bridges are formed between Asp-88(Kinase) and N2 of ring-2 and Lys-29(Kinase) with the carboxyl group of ring-3. Lys-31(Kinase) interacts with the phosphorylated serine (pSer) at position 108 from the CBM. The CBM contributes an important interaction to drug binding through Arg-83. In addition to making a hydrogen bond with Asn-48(Kinase), it also makes a cation–π stacking interaction with ring-2 of the activator. The cation–π interaction from Arg-83(CBM) with the activator is indicated by hatched lines. Additional potential interactions (where the bond lengths are longer than those expected for a high resolution structure) are indicated by a thin dashed blue line. (e) Overlay of A-769662 (cyan) and 991 (magenta) are shown in stick representation.
Figure 4
Figure 4. The structure of the α2 hook of AMPK.
(a) Ribbons representation of the overlap of the γ subunit and hook regions from full-length α2/activator complex (coloured in pink and orange) and from the ΔCBM AMPK complex (coloured in grey), showing the close structural similarity of the hook region for α1 and α2 over the five strictly conserved residues. The two exchangeable AMP moieties are shown in stick representation (from the full-length structure) but the non-exchangeable AMP has been removed for clarity. (b) Detailed view of the α2 hook (coloured in orange with the residue carbons in yellow) and its interaction with residues at the AMP-3 site on the γ-subunit (coloured pink with the residue carbons in grey) and with a loop from the β-subunit (coloured green with the carbons in grey). AMP-3 is shown in stick representation (with the carbons coloured in grey). His-366(α2-hook) forms a salt bridge with Glu-296(γ), while the following proline at position 367 introduces a kink into the chain, which seems instrumental in positioning Glu-368(α2-hook) that makes salt-bridges to Lys-170(γ) and Arg-70(γ). Arg-369(α2-hook) makes hydrogen bonds with three residues on a loop from the β-subunit, Thr-219(β), and the main-chain carbonyl oxygen of Gly-220(β). Potential interactions are indicated by a thin dashed blue line. (c) Allosteric activation of wild-type and E368A(α2-hook) α2β1γ1 complex by AMP (100 μM) (Left). In the right-hand panel, the effect of AMP (50 μM), ADP (50 μM) or 991 (100 nM) on protection against dephosphorylation for the E368A(α2-hook) α2β1γ1 complex (Right). Results are the mean ±s.e.m. for three independent experiments. (d) Schematic for the regulation of AMPK by kinase domain and CBM tethering to the regulatory fragment. The right-hand panel represents the activated full-length AMPK/991 complex reported in this paper. It consists of the regulatory fragment (R) containing the γ-subunit (in pink) and the C-termini scaffold domains of the α- and β-subunits (in light blue/green). The α2 kinase domain (phosphorylated on Thr-172 of the activation loop) is shown in yellow and is connected to the regulatory fragment by a flexible linker (in black). The interaction of the kinase domain with the regulatory fragment mainly involves the activation loop and protects Thr-172 from dephosphorylation. The CBM of the β-subunit (coloured in green) binds to the N-lobe of the kinase domain and is also connected to the regulatory fragment by a flexible linker (in black). The presence of the activator compound 991 (Act) is envisaged to strengthen the interaction between the kinase and CBM and protect a major proportion of the active enzyme against dephosphorylation. Dissociation of the activator compound gives rise to the species shown in the middle panel. In this case the enzyme becomes less active because the interaction between the CBM and the kinase domain is weaker and they therefore interact for a smaller proportion of the time. Replacing ADP (or AMP) by Mg.ATP leads to displacement of the α-hook and thus the dissociation of the kinase domain and CBM from the regulatory fragment (as shown in the left panel). In this form, the kinase is no longer allosterically activated and is susceptible to dephosphorylation, and thus inactivation.

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