Human Adenylate Kinase 5

Target ID:

AK5A

Aliases:

AK6MGC33326

Deposition Date:

Monday 18th July 2005

Families:

Non-protein Kinase

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2BWJ

Authors:

G.BUNKOCZI,P.FILIPPAKOPOULOS,O.FEDOROV,A.JANSSON,E.LONGMAN,E.UGOCHUKWU,S.KNAPP,F.VON DELFT,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,J.WEIGELT

Site:

Oxford

ICM Datapack:

ICM Datapack

2BWJ

tabs

Structure Details

Adenylate kinases (AK's) are essential for the maintenance of cellular energetic economy and are key enzymes in the synthesis and regulation of adenine nucleotides.
AK's are required for a variety of cellular metabolic processes as well as for RNA and DNA synthesis.
In general AK's catalyze the exchange of adenine nucleotide phosphate moieties (ATP + AMP 2ADP) and facilitate transfer of both Ã¢- and Ã£-phosphoryls in ATP. AK5has been shown to phosphorylate both AMP and dAMP at equal efficiency with ATP as phosphate donor, and AMP, CMP, and dCMP with GTP as phosphate donor. Thus, the substrate specificity of AK5 overlaps with that of the closely sequence-related UMP/CMP kinases.

The cellular phosphotransfer becomes particularly important in tissues with an increase energy demand.
AK's play also an important role in the activation of therapeutic nucleoside analogues used for the treatment of cancer and against virus infections.

AK5 has been identified by EST database searching for novel nucleoside monophosphate kinases. The gene encodes a 198-amino acid protein which contains a nucleoside triphosphate binding glycine-rich region, a nucleoside monophosphate-binding site, and the lid domain that closes over the substrate upon binding. Within the AK family AK5 is most closely related to AK1 58% (sequence identity). AK5 is specifically expressed in the brain and transfection experiments with an AK5-GFP fusion protein demonstrated that it is localized in the cytosol.

Materials and Methods

AK5

PDB:2BWJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_777283
Entry Clone Source:TCK
SGC Clone Accession:
Tag:TEV-cleavable (*) N-terminal his6 tag: MHHHHHHSSGVDLGTENLYFQ*S(M).
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgdlgtenlyfqsMGGFMEDLRKCKIIFIIGGPGSGKGTQCEKLVEKYGFTHLSTGELLREELASESERSKLIRDIMERGDLVPSGIVLELLKEAMVASLGDTRGFLIDGYPREVKQGEEFGRRIGDPQLVICMDCSADTMTNRLLQRSRSSLPVDDTTKTIAKRLEAYYRASIPVIAYYETKTQLHKINAEGTPEDVFLQLCTAIDSIflMutation R135M
Vector:pLIC- SGC1

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 (10 g in 100 mL of 2.5 M NaCl) was equilibrated with 100mL of Loading Buffer. A 5 mL 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 AK5A 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.

Enzymatic treatment: Treated the IMAC elution(s) with TEV protease overnight at 4°C.

Extraction

Procedure
Extraction buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol.

The cell pellets (5 g 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 600 nL sitting drops mixing 300 nL of AK5A (15 mg/mL in 10mM Hepes pH 7.5, 100mM NaCl ,10mM DTT) with 300nl of a solution containing 0.1M NaCl, 0.1M HEPES pH 7.5 and 1.6M (NH4)2SO4. Adenosine monophosphate (AMP), Adenylyl imidodiphosphate (AMPPNP) and Mg+2 were also present in the crystallization matrix at a final concentration of 1mM each. Cryo protection was achieved by adding to the crystallization mix sucrose (25% final w/v).
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Van Rompay, A. R.; Johansson, M.; Karlsson, A. (1999). Identification of a novel human adenylate kinase: cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein. Europ. J. Biochem. 261: 509-516.

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.