AMP-activated protein kinase beta 2

Target ID:

AMPKB2

Aliases:

MGC61468

Deposition Date:

Monday 14th November 2005

Families:

Protein Kinase

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2F15

Authors:

J.R.WALKER,L.WYBENGA-GROOT,P.J.FINERTY JR.,E.NEWMAN,F.M.MACKENZIE,J.WEIGELT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2F15

tabs

Structure Details

AMPK is a master regulator of energy homeostasis, integrating numerous signal transduction pathways as well as sensing levels of key intracellular energy components.
The biological roles of AMPK are complex, but can be summarized as follows:
When energy levels are down, this protein up-regulates energy-producing pathways and down-regulates energy-consuming pathways.
Its specific roles vary according to physiological context.
In peripheral tissues, low nutrient conditions, such as exercise and short-term hunger, often result in 1) increased energy production through activation of glucose-uptake and fatty acid oxidation and 2) decreased energy expenditure through inhibition of synthetic routes such as for glycogen, fatty acid, and protein (Kahn et al., 2005).
For example, AMPK-mediated phosphorylation of acetyl-CoA carboxylase inhibits the latterâ€™s catalytic activity,
which decreases production of malonyl-CoA, which increases fatty acid oxidation, and which increases ATP levels (Saha and Ruderman, 2003).
In neuronal tissues, high leptin levels associated with adiposity inhibits hypothalamic AMPK, regulating various downstream pathways that increase satiety effects on the higher brain (Minokoshi et al., 2004).

AMPK is a heterotrimeric complex composed of one subunit each of alpha (a1 and a2), beta (b1 and b2), and gamma (g1, g2, and g3). The beta and gamma subunits are regulatory components for the serine/threonine kinase domain (alpha subunit), capable of binding and sensing the concentrations of glycogen and AMP, respectively. The beta subunits have glycogen binding properties and may also act as targeting agents: by dragging AMPK to glycogen compartments, glycogen phosphorylase can be more efficiently phosphorylated and activated by AMPK (Polekhina et al., 2003).

We have determined the high resolution structure of the glycogen binding domain of the beta2 subunit of human AMPK. Two key tryptophan residues, which form one side of the putative glycogen pocket, and the sharp leucine-containing beta-turn, which forms the other side of the pocket, do not reveal major conformational changes in the absence of ligand compared with cyclodextrin-bound beta1 (Polekhina et al., 2005). But the base of the putative glycogen binding pocket contains a one residue insertion compared with beta1 that could affect substrate specificity.

Materials and Methods

AMPKB2

PDB:2F15

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:ampkb2.BC053610.MGC.AT79-C12.pCMV-SPORT6
Entry Clone Source:
SGC Clone Accession:ampkb2.070.163; plate SDC043A12
Tag:MGSSHHHHHHSSGLVPR*GS
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPR*GSVKPTQQARPTVIRWSEGGKEVFISGSFNNWSTKIPLIKSHNDFVAILDLPEGEHQYKFFVDGQWVHDPSEPVVTSQLGTINNLIHVKKSDFEVF
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 4.5. Cells were then induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at room temperature (~22 ºC). The culture was centrifuged and the cell pellets were collected and stored at -80ºC.

Purification

Procedure
IMAC purification: The clarified supernatant from a 2 L culture was loaded at approximately 1 mL/min by gravity onto 5 mL of Ni-NTA resin (Qiagen 30450). Twelve column volumes of Wash buffer were used at approximately 3 mL/min. Samples were eluted from the Ni-NTA resin by exposure to 8 mL Elution buffer at 1mL/min flow rate. A 15 µL sample of the eluate is saved for SDS-PAGE analysis. 10 µL of each eluate is saved for measurement of protein concentration using Bradford reagent (BioRad 500-0202). (His)6-tag cleavage: Thrombin (Sigma T9681; 1 unit per milligram of protein to 50 U maximum) and CaCl2 (4 mM final concentration) were added to the 8 mL of Ni-NTA eluate in 50 mL conical vials (352096, BD Biosciences) and the conical vial is stored without shaking, overnight, at 4ºC. Size exclusion chromatography: All gel filtration columns, buffers, and protocols are identical for uncut and thrombin-treated proteins. An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) is pre-equilibrated with Gel filtration buffer for 1.5 column volumes using an AKTA Express (18-6645-05, GE Healthcare) at a flow rate of 1.5 mL/min. 8 mL of sample is loaded onto the column at 1.5 mL/min, and 2 mL fractions are collected into 96-well plates (VWR 40002-012) using peak fractionation protocols with the following parameters: (Slope; min. peak width 0.833 min; level 0.000 mAU; peak start slope 10.000 AU/min; peak end slope 20.000 AU/min). Peak fractions are analyzed for purity using SDS-PAGE or visual analysis of the chromatogram and pooled. AMPKB2 eluted as multiple peaks; each peak was pooled and concentrated separately and analyzed by mass spectrometry.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 25 mL Lysis buffer and lysed using a Microfluidizer. The lysate was cleared by centrifugation and imidazole added to the clarified supernatant (5 mM final concentration).
Concentration:
Ligand
MassSpec:
Crystallization:Purified AMPKB2 was crystallized using the hanging drop vapor diffusion method. Crystals grew when the protein (5 mg/mL) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 1.6M NH4SO4, 0.2 M NaAc, 0.1 M Hepes, pH 7.5, 5% ethylene glycol in 291 K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Kahn,B.B., Alquier,T., Carling,D., and Hardie,D.G. (2005). AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism 1, 15-25.

Minokoshi,Y., Alquier,T., Furukawa,N., Kim,Y.B., Lee,A., Xue,B.Z., Mu,J., Foufelle,F., Ferre,P., Birnbaum,M.J., Stuck,B.J., and Kahn,B.B. (2004). AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428, 569-574.

Polekhina,G., Gupta,A., Michell,B.J., van Denderen,B., Murthy,S., Feil,S.C., Jennings,I.G., Campbell,D.J., Witters,L.A., Parker,M.W., Kemp,B.E., and Stapleton,D. (2003). AMPK beta subunit targets metabolic stress sensing to glycogen. Curr. Biol. 13, 867-871.

Polekhina,G., Gupta,A., van Denderen,B.J.W., Fell,S.C., Kemp,B.E., Stapleton,D., and Parker,M.W. (2005). Structural basis for glycogen recognition by AMP-activated protein kinase. Structure 13, 1453-1462.
Saha,A.K. and Ruderman,N.B. (2003). Malonyl-CoA and AMP-activated protein kinase: An expanding partnership. Molecular and Cellular Biochemistry 253, 65-70.