Human adenylate kinase 4 in complex with Diguanosine pentaphosphate

Target ID:

AK3A

Aliases:

AK3AK4

Deposition Date:

Tuesday 18th October 2005

Families:

Non-protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2BBW

Authors:

P.FILIPPAKOPOULOS,A.P.TURNBULL,O.FEDOROV,J.WEIGEL,G.BUNKOCZI,E.UGOCHUKWU,J.DEBRECZENI,F.NIESEN,F.VON DELFT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,S.KNAPP,STRUCTURALGENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2BBW

tabs

Structure Details

Adenylate kinases are essential for the maintenance of cellular energetic economy and are key enzymes in the synthesis,
equilibration and regulation of nucleotides.
AK's catalyze nucleotide exchange:
(ATP + AMP <=> 2ADP or GTP + AMP <=> GDP + ADP)
and facilitates transfer of both β- and γ-phosphoryls in ATP and other nucleotide substrates.

In contrast to cytoplasmic AKs which participate in energy metabolism when local high energy phosphate levels are low,
mitochondrial AK's (AK3 and AK4) function in the salvage pathway of AMP
because this adenine nucleotide which accumulates in periods of energy demand cannot enter the mitochondrion
and will be lost if not phosphorylated.
It is likely that both mitochondrial AK's use GTP as a substrate;
however, the substrate specificity of human AK4 as well as its role in mitochondria remains to be clarified.

The expression of AK4 mRNA is restricted in the brain hippocampus and cerebellum, olfactory neuroepithelium as well as liver.
This result indicates that in contrast to AK3, AK4 acts on the specific mechanism of unusual energy metabolism
rather than the control of homeostasis of the ADP pool ubiquitously.

It is therefore likely that the function of AK4 is rather the control of ATP/GTP metabolism
for intracellular signal transduction, production of the neurotransmitters
or the neurotrophic factors and neuroprotection in the case of ischemia.
In order to address this question we determined the structure of AK4 in complex with a possible substrate analogue (Gp5G).

AK3A

PDB:2BBW

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_013410.2
Entry Clone Source:Toronto sick kid hospital
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m). TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmASKLLRAVILGPPGSGKGTVCQRIAQNFGLQHLSSGHFLRENIKASTEVGEMAKQYIEKSLLVPDHVITRLMMSELENRRGQHWLLDGFPRTLGQAEALDKICEVDLVISLNIPFETLKDRLSRRWIHPPSGRVYNLDFNPPHVHGIDDVTGEPLVQQEDDKPEAVAARLRQYKDVAKPVIELYKSRGVLHQFSGTETNKIWPYVYTLFSNKITPIQSKEAYL
Vector:pLIC-SGC

Growth

Medium:
Antibiotics:
Procedure:Grow starter cultures from freshly transformed colonies in 10 ml LB, 0.1 mg/ml ampiciline. This started culture was diluted 1:1000 in fresh media and was grown at 37°C to an OD600 of 0.4 and than transferred to 18°C. Expression was induced at an OD600 of 0.6 - 0.7 using 1 mM IPTG (final concentration). Cells were harvested after 4h by centrifugation (15min, 6000rpm on a JLA 8.100 rotor), transferred to 50-ml tubes, and frozen at -20°C.

Purification

Procedure
Column 1: A DE52 column (10gr in 100ml of 2.5M NaCl) was equilibrated with 100ml of Loading Buffer. A 5ml NiNTA column was equilibrated with 20ml of Loading Buffer

Buffers: Loading buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 5% glycerol. Wash buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% glycerol. Elution buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 50-250 mM imidazole (step elution), 5% glycerol.

Procedure: A DE52 column (10gr suspended in 100ml of 2.5M NaCl) was equilibrated with 100ml of Loading Buffer. A 5ml NiNTA column was equilibrated with 20ml of loading buffer. The lysed sample was applied to the DE-52 column and washed through with 50 ml loading buffer. The flow through was applied to the 5 ml Ni-NTA column which was washed with 2x10ml of wash buffer and eluted with elution buffer in 5 ml aliquots. (Step elution using 50, 100, 150, 200 and 250 mM imidazole in the Elution Buffer)

Column 2: SEC

Buffers: Gel Filtration Buffer: 10mM HEPES, pH 7.5, 100mM NaCl

Procedure: AKTA-prime. Fractions containing AK4 collected from IMAC and treated with TEV protease overnight (identified by SDS PAGE) were concentrated to about 1.5ml and directly applied to a S75 16/60 column equilibrated in 10 mM Hepes pH 7.5, 100 m NaCl. The flow rate was 1ml/min and the pure protein eluted at 60-70min.

Extraction

Procedure
50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol. The cell pellets (5 gr wet wt) were re-suspended in 50 ml extraction buffer containing a Protease Inhibitor cocktail tablet (Roche), and lysed in a high pressure cell disrupter. The supernatant was centrifuged for 30 minutes at 35k g in a JA 25.5 rotor at 4°C.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown at 4°C in 200nl sitting drops mixing 50nl of AK4 (15 mg/ml in 10mM Hepes pH 7.5, 100mM NaCl ,10mM DTT containing 1 mM Gp5G) with 150nl of a solution containing 0.7M sodium succinate. Cryo protection was achieved by adding to the crystallization mix sucrose 1.2 M sodium succinate solution.
NMR Spectroscopy:
Data Collection:Resoultion: 2.05 Å; X-ray source: rotating anode (Rigaku FR-E SuperBright), single wavelength.
Data Processing:

References

Yoneda T , Sato M , Maeda M , Takagi H . (1998). Identification of a novel adenylate kinase system in the brain: cloning of the fourth adenylate kinase. Brain Res Mol Brain Res. 62(2):187-195.

Noma T , Fujisawa K , Yamashiro Y , Shinohara M , Nakazawa A , Gondo T , Ishihara T , Yoshinobu K . (2001). Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix. Biochem J. 358 :225-232.

Schulz GE, Schiltz E, Tomasselli AG, Frank R, Brune M, Wittinghofer A, Schirmer RH. (1986). Structural relationships in the adenylate kinase family. Eur J Biochem. 161, 127-32.