Human carbonic anhydrase VIII

Target ID:

CA8A

Aliases:

CA-VIIICA8CALSCARPMGC120502MGC99509

Deposition Date:

Friday 31st October 2008

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

2W2J

Authors:

Kramm, A., Muniz, J.R.C., Picaud, S.S., von Delft, F., Pilka, E.S., Yue, W.W., King, O.N.F., Kochan, G., Pike, A.C.W., Filippakopoulos, P., Arrowsmith, C.H., Wikstrom, M., Edwards, A., Bountra, C., Oppermann, U.

Site:

Oxford

ICM Datapack:

ICM Datapack

2W2J

tabs

Structure Details

The carbonic anhydrase (CA) family of enzymes catalyzes the Zn-dependent hydration of carbon dioxide to form bicarbonate and proton ions. They are ubiquitous in prokaryotes and eukaryotes, and are encoded by 4 evolutionarily-unrelated gene families (α-, β-, γ-, δ-). In humans 12 active α-isoforms exist with different tissue distributions, sub-cellular localization and kinetic properties. These include the cytosolic forms (CA I, CA II, CA III, CA VII, CA XIII), membrane-bound forms (CA IV, CA IX, CA XII, CA XIV), mitochondrial forms (CA VA, CA VB) and a secreted form (CA VI). They have an absolute requirement for a divalent Zn2+ ion in the active site, coordinated by three invariant histidine residues and a bound hydroxide ion.

CAs participate in many physiological and pathological processes such as CO2 and pH homeostasis, bone resorption, biosynthesis (e.g. gluconeogenesis, ureagenesis), and tumourigenicity. Sulphonamide-based CA inhibitors such as acetazolamide, methazolamide and ethoxzolamide are established diuretics and anti-glaucoma drugs, and may have further potentials in anti-obesity and anti-cancer treatment.

The human CA family also includes 3 non-catalytic isoforms (CA VIII, X, XI), known as CA-related proteins (CA-RPs). They lack one or more of the essential Zn-coordinating histidines and are devoid of CO2 hydration activity. The first identified CA-RP, CA VIII, is abundant in the cerebellum, and a mouse gene deletion causes a motor coordination defect. The protein is over-expressed in colon and nonsmall cell lung carcinomas, although its biological functions are unknown.

Materials and Methods

Entry Clone Source: Ordered-synthetic

Entry Clone Accession: n/a

SGC Construct ID: CA8A-c010

GenBank GI number: gi|22027500

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence: N-terminal His-tag with a TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq(*)sm

Tag removed: yes

Expressed sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqsmADLSFI
EDTVAFPEKEEDEEEEEEGVEWGYEEGVE
WGLVFPDANGEYQSPINLNSREARYDPSL
LDVRLSPNYVVCRDCEVTNDGHTIQVILK
SKSVLSGGPLPQGHEFELYEVRFHWGREN
QRGSEHTVNFKAFPMELHLIHWNSTLFGS
IDEAVGKPHGIAIIALFVQIGKEHVGLKA
VTEILQDIQYKGKSKTIPCFNPNTLLPDP
LLRDYWVYEGSLTIPPCSEGVTWILFRYP
LTISQLQIEEFRRLRTHVKGAELVEGCDG
ILGDNFRPTQPLSDRVIRAAFQ

Host: BL21(DE3)-R3-pRARE2

Expression protocol: Competent BL-21 (DE3)-R3-pRARE2 phage resistant cells were transformed with 6 µl of the plasmid DNA and plated out onto LB plate plus 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. The next day colonies were picked into fresh deep well blocks containing 1 ml TB + 50 µg/ml kanamycin and 35 µg/ml chloramphenicol which were grown overnight and glycerol stocks were prepared by adding 333 µl of 60 % glycerol to 1 ml of cell suspension, which were stored at -80°C to be used for future scale up preparations. The glycerol stock 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 the same medium. 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 CA8Awas expressed at that temperature over night. The next day cells were harvested by centrifugation at 4000 rpm for 20 minutes then the pellets were scraped out and transferred to 50-ml Falcon tubes and frozen at -80°C.

Cell extraction: 2x Lysis buffer: 100 mM K-phosphate, pH 7.5, 1M NaCl, 1 mM TCEP, 1x Protease Inhibitors Cocktail Set VII (Calbiochem, 1/1000 dilution), and 15 units/ml Benzonase. Lysis buffer: 50 mM K-phosphate, pH 7.5, 0.5M NaCl, 1 mM TCEP.

Procedure: Frozen cell pellets (g) were thawed briefly in a bath of warm water (37°C) 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 150 ml. The cells were resuspended by agitating and disrupted by high pressure homogenization (30 kpsi). Nucleic acids and cell debris were removed by adding 0.15% PEI (polyethyleneimine) from a 5% (w/v, pH 7.5) stock, stirring for 30 minutes, then centrifugation for 1 hour at 16,000 x g. The supernatant was then further clarified by filtration (Acrodisc filters, 0.2 mm).

Column 1: Ni-affinity, HisTrap Crude FF, 5 ml (GE Healthcare)

Buffers: Affinity buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP; Wash buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 30 mM imidazole, 0.5 mM TCEP; Elution buffer: 50 mM K-phosphate, pH 7.5, 500 mM NaCl, 300 mM imidazole, 0.5 mM TCEP.

Procedure: The cell extract was loaded on the column at 4 ml/minute on an AKTA-express system (GE Healthcare). The column was washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 4 ml/min. The eluted peak of A280 was automatically collected.

Column 2: Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare)

GF buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP, 4 mM MgCl2.

Procedure: The eluted fraction from the Ni-affinity Histrap column was loaded on the gel filtration column in GF buffer at 0.80 ml/min. Eluted proteins were collected in 2-ml fractions and analyzed on SDS-PAGE.

Mass spectrometry characterization: ESI-MS revealed that the protein had a mass of 33060.3242 Da (expected mass: 33060).

Compound: none.

Protein concentration: Buffer was changed to 10 mM HEPES, 150 mM NaCl, 5% glycerol. The protein was concentrated to 10.5 mg/ml using a centricon with a 10kDa cut off. The protein concentration was determined spectrophotometrically using e280= 46410.

Crystallization: Crystals were grown at 4°C by vapour diffusion in sitting drops mixing 100 nL protein (10.5 mg/mL) and 200 nL well solution containing 10% (w/v) PEG 6000, 0.3 M NH₄Cl pH 6.3 and 10% (v/v) ethylene glycol. Crystals were cryo-protected using 20% (v/v) ethylene glycol and flash cooled in liquid nitrogen.

Data Collection: Resolution :1.6Å; X-ray source: SLS beam 10XSA