Human adenosine deaminase

Target ID:

ADAA

Aliases:

ADA

Deposition Date:

Tuesday 14th July 2009

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

protein-ligand

Download Coordinates:

3IAR

Authors:

Ugochukwu, E., Zhang, Y., Hapka, E., Yue, W.W., Bray, J.E., Muniz, J., Burgess-Brown, N., Chaikuad, A., von Delft, F., Bountra, C., Arrowsmith, C.H., Weigelt, J., Edwards, A., Oppermann, U.

Site:

Oxford

3IAR

tabs

Structure Details

Adenosine deaminase (ADA) is part of the purine catabolic pathway and catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxyinosine respectively. There are currently two main objectives of ADA research. Firstly, mutations in the ADA gene cause severe combined immunodeficiency (ADASCID), a fatal condition if left untreated. Secondly, there is interest in developing pharmacological agents to inhibit ADA that would increase extracellular adenosine concentrations and reduce tissue damage.

The rare genetic disorder ADASCID was the first to be treated by enzyme replacement therapy and more recently there has been success with gene replacement therapy. ADASCID is an autosomal recessive disorder characterized by missing lymphocytes and susceptibility to persistent infection. There are 29 known genotypes associated with the disorder. Bone marrow transplantation is highly successful if a matched sibling or related donor is available. For individuals without a matched donor, administration of a PEG-modified bovine ADA has been in use since the late 1980s. Enzyme replacement therapy with PEG-ADA is generally well-tolerated although the treatment is costly and the patients must take the drug their entire lives. Early clinical trials of ADA gene therapy used methods that are now considered to be suboptimal. These patients continued to receive PEG-ADA and showed no significant benefit over enzyme replacement therapy alone, although no serious adverse affects were observed. A subsequent study, which eliminated PEG-ADA therapy and included chemotherapy to reduce marrow stem cell populations prior to introducing CD34+ cells transduced with retroviral ADA, has demonstrated a positive immune system restoration. Although this is a relatively new therapy the success rate indicates it should be considered for ADASCID patients who lack a matched sibling donor.

Extracellular adenosine, acting through G-protein coupled receptors, has protective cardiovascular properties such as reducing inflammation and promoting vasodilation. Therefore, use of drugs that affect adenosine metabolism could be beneficial. Although normally metabolized in the cytosol where it is irreversibly deaminated by ADA or rephosphorylated by adenosine kinase, during ischemia or hypoxia extracellular deamination of adenosine is the main pathway of its degradation. Animal studies have long shown the tissue-protective benefits of adenosine but translating this knowledge into clinical therapies has been problematic. One difficulty is measuring the adenosine concentration in plasma, where it has a half-life of less than 1 s and continues to be degraded after blood withdrawal. Additionally, there is concern that systemwide administration of ADA inhibitors could produce undesirable side effects. Nevertheless, it is likely that some of the anti-inflammatory properties of the drug methotrexate are due to AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) accumulation and inhibition of ADA. Other drugs in current use that are known to inhibit ADA activity include lidoflazine, dipyridamole, trazodone, and phenylbutazone.

ADA is a (β/α)8 barrel enzyme with a catalytic zinc atom located at the C-terminal end of the barrel. In this structure the zinc is replaced by a nickel atom that is coordinated by 3 histidines, an aspartate, and the deoxyadenosine N6.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:3629376

SGC Construct ID: ADAA-c103

GenBank GI number: gi|47078295

Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CTTAAGAAGGAGATATACTATGCCCGCCT
TCGACAAGCCCAAAGTAGAACTGCATGTC
CACCTAGACGGATCCATCAAGCCTGAAAC
CATCTTATACTATGGCAGGAGGAGAGGGA
TCGCCCTCCCAGCTAACACAGCAGAGGGG
CTGCTGAACGTCATTGGCATGGACAAGCC
GCTCACCCTTCCAGACTTCCTGGCCAAGT
TTGACTACTACATGCCTGCTATCGCGGGC
TGCCGGGAGGCTATCAAAAGGATCGCCTA
TGAGTTTGTAGAGATGAAGGCCAAAGAGG
GCGTGGTGTATGTGGAGGTGCGGTACAGT
CCGCACCTGCTGGCCAACTCCAAAGTGGA
GCCAATCCCCTGGAACCAGGCTGAAGGGG
ACCTCACCCCAGACGAGGTGGTGGCCCTA
GTGGGCCAGGGCCTGCAGGAGGGGGAGCG
AGACTTCGGGGTCAAGGCCCGGTCCATCC
TGTGCTGCATGCGCCACCAGCCCAACTGG
TCCCCCAAGGTGGTGGAGCTGTGTAAGAA
GTACCAGCAGCAGACCGTGGTAGCCATTG
ACCTGGCTGGAGATGAGACCATCCCAGGA
AGCAGCCTCTTGCCTGGACATGTCCAGGC
CTACCAGGAGGCTGTGAAGAGCGGCATTC
ACCGTACTGTCCACGCCGGGGAGGTGGGC
TCGGCCGAAGTAGTAAAAGAGGCTGTGGA
CATACTCAAGACAGAGCGGCTGGGACACG
GCTACCACACCCTGGAAGACCAGGCCCTT
TATAACAGGCTGCGGCAGGAAAACATGCA
CTTCGAGATCTGCCCCTGGTCCAGCTACC
TCACTGGTGCCTGGAAGCCGGACACGGAG
CATGCAGTCATTCGGCTCAAAAATGACCA
GGCTAACTACTCGCTCAACACAGATGACC
CGCTCATCTTCAAGTCCACCCTGGACACT
GATTACCAGATGACCAAACGGGACATGGG
CTTTACTGAAGAGGAGTTTAAAAGGCTGA
ACATCAATGCGGCCAAATCTAGTTTCCTC
CCAGAAGATGAAAAGAGGGAGCTTCTCGA
CCTGCTCTATAAAGCCTATGGGATGCCAC
CTTCAGCCTCTGCAGGGCAGAACCTCGCA
GAGAACCTCTACTTCCAATCGCACCATCA
TCACCACCATGATTACAAGGATGACGACG
ATAAGTGAGGATCC

Tags and additions: C-terminal His6 tag and FLAG tag, preceded by a TEV protease cleavage site:
aenlyfq*shhhhhhdykddddk (* - TEV cleavage site)

Expressed sequence (tag sequence in lowercase):
MPAFDKPKVELHVHLDGSIKPETILYYGR
MPAFDKPKVELHVHLDGSIKPETILYYGR
RRGIALPANTAEGLLNVIGMDKPLTLPDF
LAKFDYYMPAIAGCREAIKRIAYEFVEMK
AKEGVVYVEVRYSPHLLANSKVEPIPWNQ
AEGDLTPDEVVALVGQGLQEGERDFGVKA
RSILCCMRHQPNWSPKVVELCKKYQQQTV
VAIDLAGDETIPGSSLLPGHVQAYQEAVK
SGIHRTVHAGEVGSAEVVKEAVDILKTER
LGHGYHTLEDQALYNRLRQENMHFEICPW
SSYLTGAWKPDTEHAVIRLKNDQANYSLN
TDDPLIFKSTLDTDYQMTKRDMGFTEEEF
KRLNINAAKSSFLPEDEKRELLDLLYKAY
GMPPSASAGQNLaenlyfqshhhhhhdyk
ddddk

Tag removed: yes

Host: BL21(DE3)-R3-pRARE2

Expression protocol: A glycerol stock of host strain BL21(DE3)-R3-pRARE2 carrying the expression plasmid was used to inoculate 10 ml of TB (terrific Broth) supplemented with 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate a 1 liter culture in TB supplemented with 50 µg/ml kanamycin only. The culture was grown at 37°C until the OD₆₀₀ reached ˜3.0. After that the temperature was lowered to 18°C. Protein production was induced with 0.1 mM IPTG and recombinant ADAA was expressed at that temperature overnight. The next day cells were harvested by centrifugation at 5000 rpm for 20 minutes and pellets were frozen at -80°C.

Cell extraction:
2x Lysis buffer: 100 mM HEPES, pH 7.5, 1 M NaCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP, 2x Protease Inhibitors Cocktail Set VII (Calbiochem, 1/1000 dilution), Benzonase (3 µl per 25 ml, 30 units/ml).
Procedure:
Frozen cell pellets were thawed at room temperature for approximately 1 hour then transferred to ice. One volume (i.e. 1 ml for every gram of cells) of 2x lysis buffer was added, followed by 1x lysis buffer to a total volume of 40 ml. The cells were resuspended by agitating and disrupted by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI (polyethyleneimine) from a 5% (w/v) stock, then centrifuged for 60 minutes at 16,000 rpm. The supernatant was further clarified by filtration (Acrodisc filters, 0.2 µm).
Column 1: 5-ml HisTrap Crude FF column (GE Healthcare)
Column 2: Gel filtration, Hiload 16/60 Superdex S200 prep grade, 120 ml (GE Healthcare)

Solutions:
Lysis buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% glycerol, 0.5 mM TCEP.
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% glycerol, 0.5 mM TCEP.
Elution buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% glycerol, 0.5 mM TCEP.
Gel Filtration Buffer - 10mM HEPES pH 7.5, 500mM NaCl, 5% glycerol and 0.5 mM TCEP

Procedure: The clarified supernatant was loaded on a 5-ml HisTrap Crude FF column at 4 ml/min using an AKTAxpress system. The column was washed with wash buffer containing 30 mM imidazole, and eluted with elution buffer containing 300 mM imidazole. The eluted fraction was collected automatically and further fractionated on a S200 HR 16/60 gel filtration column equilibrated with gel filtration buffer. Fractions containing ADAA protein were identified by SDS-PAGE, combined, and concentrated to 15 mg/ml using centrifugation (5 kDa MWCO, Centricon). Concentrated protein samples were flash frozen in liquid nitrogen and stored at -80 °C

Enzymatic treatment: His-tagged TEV protease was added to the protein using a 1:20 TEV to protein ratio (mg/mg). It was incubated overnight at 4°C.

Column 3:Ni-NTA (Qiagen)

Buffers:
Wash buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% glycerol, 0.5 mM TCEP.
Elution buffer: 50 mM HEPES, pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% glycerol, 0.5 mM TCEP.

Procedure: Following TEV digestion, the protein sample was passed through 1ml Ni-NTA in a 10mm gravity flow column and the flow-through collected. The column was washed with 10 ml of wash buffer and the wash and flow-through fractions were analyzed by SDS-PAGE and pooled.

Mass spectrometry characterization : ESI-MS revealed that the protein had a mass of 41200Da (Expected mass 41330Da), the -130Da mass difference was attributed to loss of the initiator methionine.

Compound: Adenine deoxyriboside (deoxyadenosine)

Protein concentration: Protein stored at -80°C was thawed and diluted with buffer (10 mM HEPES pH7.5, 150 mM NaCl). A 2 to 3 times molar excess of compound was mixed with the protein and the solution was concentrated to 25 mg/ml using a Centricon filtration unit with a 5kDa MW cut-off.

Crystallization: Crystals were grown at 20°C by vapour diffusion in sitting drops by mixing protein (25 mg/ml) and well solution containing 20% PEG 3350 and 0.20M NaNO₃ at a protein to precipitant ratio of 1:2. A crystal was cryo-protected using well solution supplemented with 25% (v/v) glycerol and flash-cooled in liquid nitrogen.

Data Collection: Resolution (scaled): 1.6 Å; X-ray source: rotating anode (Rigaku FR-E SuperBright)

References

Aiuti, A., F. Cattaneo, et al. (2009). "Gene therapy for immunodeficiency due to adenosine deaminase deficiency." N Engl J Med 360(5): 447-58.

Cristalli, G., S. Costanzi, et al. (2001). "Adenosine deaminase: functional implications and different classes of inhibitors." Med Res Rev 21(2): 105-28.

Riksen, N. P., G. A. Rongen, et al. (2008). "Human in vivo research on the vascular effects of adenosine." Eur J Pharmacol 585(2-3): 220-7.