Human AMP-activated protein kinase alpha 2

Target ID:

AMPKA2

Aliases:

AMPKAMPK2PRKAA

Deposition Date:

Wednesday 31st May 2006

Families:

Protein Kinase

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2H6D

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2H6D

tabs

Structure Details

AMPK is a master regulator of metabolism and thus controls the energy status at a cellular level. It is increasingly becoming evident that AMPK is also involved in energy regulation at the level of the whole organism through its response to hormonal cues and nutrient availability[1,2]. Interest in this area has been sparked by the discovery that some AMPK isoforms are indirectly activated by leptin, a hormone that regulates food-intake, and by metformin and thiazolidinediones, both currently popular treatments for Type 2 diabetes[2].

The energy status of a cell is intimately associated with the ratio in the concentration of ATP and ADP (or AMP). AMPK senses changes in the AMP:ATP ratio, being activated simultaneously by falling concentrations of ATP and rising concentrations of AMP. In response, it acts to shut down ATP consuming pathways and activate ATP producing pathways. AMPK phosphorylates the bottleneck enzymes in these pathways. Phosphorylation down-regulates those involved in anabolism and up-regulates those involved in catabolism, restoring the ATP:ADP balance.

In the basal state, AMPK activity is very low. Activation of AMPK includes phosphorylation of its activation loop threonine by an upstream kinase as well as γ-subunit-mediated release of the α-subunit autoinhibitory region. There are multiple isoforms of each subunit in the human genome including two isoforms of the α and β subunits (α1, α2, β1 and β2) and three of the γ (γ1, γ2 and γ3)[3]. We have solved the minimal-kinase domain of the α2 subunit at 1.9Ã… resolution. The structure reveals the canonical kinase fold with an interesting conformation by its activation loop. Notably, the conserved and catalytically important DFG motif, which is located immediately N-terminal to the activation loop, is flipped out and inserted into the ATP binding pocket. In addition, the amino-terminal lobe clamps down tightly against the DFG motif, mimicking the closed lobe orientation seen in certain other kinases. This may represent a mechanism of auto-inhibition used by AMPK.

Materials and Methods

AMPKA2

PDB:2H6D

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_006243
Entry Clone Source:ampka2.006.279; plate SDC062:G8
SGC Clone Accession:
Tag:mgsshhhhhhssglvprGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:gsKHDGRVKIGHYVLGDTLGVGTFGKVKIGEHQLTGHKVAVKILNRQKIRSLDVVGKIKREIQNLKLFRHPHIIKLYQVISTPTDFFMVMEYVSGGELFDYICKHGRVEEMEARRLFQQILSAVDYCHRHMVVHRDLKPENVLLDAHMNAKIADFGLSNMMSDGEFLRTSCGSPNYAAPEVISGRLYAGPEVDIWSCGVILYALLCGTLPFDDEHVPTLFKKIRGGVFYIPEYLNRSVATLLMHMLQVDPLKRATIKDIREHEWFKQDLPSYLFPE
Vector:p28a-thrombin-lic

Growth

Medium:Terrific Broth (TB)
Antibiotics:
Procedure:The host was grown in Terrific Broth (TB) in the presence of kanamycin (50 µg/mL). A stab culture was taken from glycerol stocks and inoculated into 100mL of LB with 50ug/mL kanamycin in a 250mL flask and incubated with shaking at 250rpm overnight at 37 degC. The culture was transfered into 1.8L TB with 50ug/mL kanamycin and 0.6mL of antiforam (Sigma) in 2L bottles and cultured using the LEX system to an OD600 of 4.0-5.0 before induction. The culture was adjusted to 15degC and isopropyl-1-thio-D-galactopyranoside (IPTG) was added to 100ug/mL then incubated overnight at 15degC before harvesting by centrifugation and storage at -80degC.

Purification

Procedure
Imidazole was added to the cleared cell lysate to bring it to a final concentration of 5mM imidazole before loading onto 4 mL bed-volume of Ni-NTA agarose affinity resin (Qiagen #30250). The column was then washed with 50mL of binding buffer and 50mL of Wash buffer.The Ni-NTA resin was equilibrated in the thrombin cleavage buffer by washing 5-10mL over the resin. After this elution, the stopcock valve was closed. A 10mL volume of thrombin cleavage buffer and 20-40 Units of bovine plasma thrombin was added to the resin. The beads were rocked for 16hours at either 4 degrees or room temperature.The eluate was loaded onto an an XK 16x65 column packed with HighLoad Superdex 200 resin that was pre-equilibrated in gel filtration buffer (AKTAxpress, GE Healthcare). The gel filtration column was run at a flow rate of 1.5mL/min and 2mL fractions collected. Fractions were pooled and concentrated using a centrifugal filter device (Amicon YM10).

Extraction

Procedure
The cell-pellet was thawed, then resuspended in 50mL of binding buffer containing Sigma\'s protease inhibitor cocktail (P2714-1BTL). The thawed cells were homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds. The cells were then lysed by sonication (Virtis408912, Virsonic) on ice with a 10 seconds pulse at half-maximal frequency then 10 seconds rest for a total of 5 minutes sonication time. The resulting lysate was then centrifuged at 63000 xg for 30 minutes at 10 degC.
Concentration:
Ligand
MassSpec:
Crystallization:The purified protein was crystallized using a combination of sitting and hanging drop vapor diffusion methods. Protein was mixed with an equal volume of reservoir solution, and the plate incubated at 4 or 18 degC. Crystals were appropriately cryoprotected as indicated below and harvested into liquid nitrogen for data collection.Reservoir: 18.6% PEG 4000, 0.1M AmSO4, 0.1M Tris-HCl pH 8.5, 15%v/v isopropanol, 5mM ATP/MgCl; Cryo: 25% w/v glycerol
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Management of Cellular energy by the AMP-activated protein kinase system
Hardie D. G., Scott J. W., Pan D. A. and Hudson E. R.
FEBS Letters 546 pp 113-120 (2003)
AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism.
Kahn B. B., Alquier T., Carling D. and Hardie G. D.
Cell Metabolism Jan. Vol 1. (2005)
Functional Domains of the α1 Catalytic Subunit of the AMP-activated Protein Kinase
Crute B. E., Seefeld K., Gamble J., Kemp B. E. and Witters L. A.
J. Biol. Chem. 273 (52) pp. 35347-35354 (1998)