Human adenylate kinase

Target ID:

AK1A

Deposition Date:

Friday 9th December 2005

Families:

Non-protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2C95

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2C95

tabs

Structure Details

Adenylate kinase 1 is essential for the maintenance of cellular energetic economy and is a key enzyme in the synthesis, equilibration and regulation of adenine nucleotides. AK1 catalyzes adenine nucleotide exchange (ATP + AMP <-> 2ADP) and facilitates transfer of both β- and γ-phosphoryls in ATP. The cellular phosphotransfer becomes particularly important during in tissues with an increase energy demand. Like it has been shown that in heart failure AK1 expression levels correlate well with functional recovery in the metabolically compromised heart. Isoforms of adenylate kinase are found in mitochondria, cytosol, and cellular membranes, creating an integrated phosphotransfer network. An intimate relationship of adenylate kinase with mitochondrial respiration, myofibrillar and membrane ATPases, as well as metabolic sensors such as ATP-sensitive K+ channels, contributes to efficient regulation of energy metabolism, membrane excitability, and cell contraction. The membrane bound form, AK1 beta, is a specific isoform, which has a presumed role in compartmentalized ATP-GTP homeostasis and a growth-regulatory function.

Genetic deletion of AK1 reduces the cellular energetic economy and increases cell vulnerability to injury. In humans, adenylate kinase deficiency caused by mutations in the AK1 gene has been associated with severe hemolytic anemia and psychomotor impairment. Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. Several mutations have been described ranging from amino acid substitutions, missense mutations and deletions allele, which resulted in nonsperocytic hemolytic anemia.

AK1 knockout mice have a decreased potential of myofibers to sustain nucleotide ratios despite upregulation of high energy phosphoryl flux through glycolytic guanolyte and creatine kinase phosphotransfer pathways. They also show decreased tolerance to metabolic stress in heart and skeletal muscle. In addition, decreased AK1 activity has been found in a mouse model for muscular dystrophy (mdx mice) suggesting a direct relationship between the lack of dystrophin and alteration of AK1.

Materials and Methods

Entry Clone Source: TKC

Entry Clone Accession: n/a

SGC Construct ID: AK1A-c001

GenBank GI number: gi|4502011

Final protein sequence:

mhhhhhhssgvdlgtenlyfqSMEEKLKKTNIIFV
VGGPGSGKGTQCEKIVQKYGYTHLSTGDLLRSEVS
SGSARGKKLSEIMEKGQLVPLETVLDMLRDAMVAK
VNTSKGFLIDGYPREVQQGEEFERRIGQPTLLLYV
DAGPETMTQRLLKRGETSGRVDDNEETIKKRLETY
YKATEPVIAFYEKRGIVRKVNAEGSVDSVFSQVCT
HLDALLN

Vector: pLIC- SGC1. Details [PDF]; Sequence [FASTA] or [GenBank]

Tags and additions: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Host: BL21 (DE3)

Growth medium, induction protocol: Grow starter cultures from freshly transformed colonies in 10 ml LB, 0.1 mg/ml amp. This started culture was diluted 1:1000 in fresh media and was grown at 37°C to a OD₆₀₀ of 0.3 and than transferred to 18°C. Expression was induced at an OD₆₀₀ of 0.6 using 1 mM IPTG. Cells were harvested after 4h by centrifugation, transferred to 50-ml tubes, and frozen in -20oC.

Extraction buffer, extraction method: Extraction buffer: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl. The cell pellets (4 gr wet wt) were re-suspended in 50 ml extraction buffer containing a Protease Inhibitor Coctail tablet (Roche), and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 17 rpm in a JA 25.5 rotor

Column 1 : DE52/Ni-NTA

Buffers: Loading buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol. Wash buffer: 50 mM Hepes, pH 7.5, 500 mM NaCl, 20 mM imidazole, 5% glycerol. Elution buffer: 50 mM Hepesl, pH 7.5, 500 mM NaCl, 50-250 mM imidazole, 5% glycerol.

Procedure: Gravity feed chromatography. Sample applied to a 10 ml DE-52 column and washed through with 50 ml loading buffer. The flow through was applied to a 1 ml Ni-NTA column, the Ni-NTA column 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)

Enzymatic treatment: Treated the IMAC elution(s) with TEV protease overnight.

Column 2: SEC

Procedure : AKTA-prime. Fractions containing AK1A collected from IMAC and treated with TEV protease overnight were concentrated to 1.5ml and directly applied to a S75 16/60 column equilibrated in 10 mM Hepes pH 7.5, 100 m NaCl. Flow rate 1ml/min.

Mass spec characterization: LC- ESI -MStof confirmed the correct mass expected for this construct.

Protein concentration: Centricons 10 kDa cut off

Crystallization: 1Z83: Crystals were grown at 4°C in 500nl sitting drops mixing 250 nl of AK1 (14 mg/ml in 10mM Hepes pH 7.5, 100mM NaCl, 10mM DTT) with 250 nl of a solution containing 20% PEG 550, 0.005 M ZnSO₄, MES pH 6.5 and 1mM AP 5 A (Diadenosine pentaphosphate pentalithium salt - CAS # 94108-02-8). 2C95: Crystal were obtained at 4 °C in sitting drops by mixing 100 nl AK1 [20 mg/ml, in 10mM HEPES pH=7.5, 100mM NaCl, 10mM DTT, 1 mM ZnSO₄, 1 mM AP4A (diadenosine tetraphosphate) ] with 50 nl of the reservoir solution 2.4 M Na-malonate pH=7.0.

Data Collection: 1Z83: Resolution: 1.9Å, X-ray source: SLS X06SA, single wavelength. 2C95: Resolution: 1.7Å, X-ray source: rotating anode (Rigaku FR-E SuperBright), single wavelength.

References

Bianchi, P, Zappa M, Bredi E, Vercellati, and Pelissero G. (199). A case of complete adenylate kinase deficiency due to a nonsense mutation in AK-1 gene (Arg 107 Stop, CGA TGA) associated with chronic haemolytic anaemia. Br J Haematol 105, 75-79.

Carrasco, AJ, Dzeja PP, Alekseev AE, Pucar D, Zingman LV, Abraham MR, Hodgson D, Bienengraeber M, Puceat M, Janssen E, Wieringa B, and Terzic A. (2001). Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc Natl Acad Sci 98, 7623-7628.

Dzeja, PP, and Terzic A. Phosphotransfer reactions in the regulation of ATP-sensitive K + channels. (1998). FASEB J 12: 523-529.

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.