Human carbonic anhydrase VI

Target ID:

CA6A

Aliases:

CA6GUSTINMGC21256

Deposition Date:

Thursday 27th November 2008

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

3FE4

Authors:

Pilka, E.S., Kochan, G., Krysztofinska, E., Muniz, J., Yue, W.W., Roos, A.K., von Delft, F., Arrowsmith, C.H., Weigelt, J., Edwards, A., Bountra, C., Oppermann, U.

Site:

Oxford

ICM Datapack:

ICM Datapack

3FE4

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 15 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), secreted form (CA VI) and acatalytic proteins (CA VIII, CA X, CA XI).

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.

CAVI is the only secreted isozyme in the mammalian CA family. It is a 42kDa glycoprotein found in the secretions of nasal, lacrimal, von Ebner’s and mammary glands of all mammals. It has also been recently identified in the enamel organ cells of rodent teeth.

Materials and Methods

Entry Clone Source: Ordered-synthetic

Entry Clone Accession: n/a

SGC Construct ID: CA6A-c110

GenBank GI number: gi|70167127

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

Tags and additions: C-terminal, TEV cleavable hexahistidine tag. Tag sequence: enlyfq(*)shhhhhhdykddddk

Host:E. coli BL21(DE3)-R3

Sequence (tag sequence in lowercase):
MSDWTYSEGALDEAHWPQHYPACGGQRQS
PINLQRTKVRYNPSLKGLNMTGYETQAGE
FPMVNNGHTVQISLPSTMRMTVADGTVYI
AQQMHFHWGGASSEISGSEHTVDGIRHVI
EIHIVHYNSKYKSYDIAQDAPDGLAVLAA
FVEVKNYPENTYYSNFISHLANIKYPGQR
TTLTGLDVQDMLPRNLQHYYTYHGSLTTP
PCTENVHWFVLADFVKLSRTQVWKLENSL
LDHRNKTIHNDYRRTQPLNHRVVESNFPN
QEYTLGSEFQAenlyfq

enlyfq residues originate from the vector and remain after the TEV cleavage of the hexahistidine tag.

Expression: 10 µl of BL21(DE3)-R3 glycerol stock were inoculated into 100ml of TB with 50µg/ml of kanamycin and 34µg/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 1L of TB with 50µg/ml kanamycin and incubated at 37°C, 160rpm. After the OD₆₀₀ reached 1.0, the temperature was dropped to 18°C and 500µl of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18°C, 160rpm. The following morning the 4L culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 80ml of a lysis buffer and frozen at -80°C.

Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml). The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45 min at 15.000rpm.

Purification:
Column 1: Ni-affinity, His-Trap, 1 ml (Amersham)
Column 2: Superdex 200, HiPrep 16/60 (Amersham)

Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol, EDTA-free Complete, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 40mM Imidazole, 5% glycerol, EDTA-free Complete, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole, 0.5mM TCEP; GF buffer: 10mM HEPES pH 7.5, 150mM NaCl, 5% glycerol, 0.5mM TCEP.

Procedure: The cell extract was loaded on the AKTA Express system, extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage.

TEV cleavage: The His-tag was cleaved with 1mg TEV per 40 mg target protein at 4°C overnight. The protein was purified on IMAC Sepharose using buffers as above. Protein was characterised by ESI-TOF mass spectrometry.

Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 10 kDa cutoff, the sample was buffer-exchanged into GF buffer and concentrated to 9mg/ml. Concentrations were determined from the absorbance at 280 nm using a NanoDrop spectrophotometer.

Mass spectrometry characterization: Calculated mass of the construct was 31914. Experimental mass determination indicates loss of the N-terminal Met residue, however a yet undefined loss of 35 Da is observed.

Crystallisation: Crystals were grown by vapor diffusion at 4°C in 150nl sitting drops. The drops were prepared by mixing 50nl of protein solution and 100nl of precipitant consisting of 0.16M MgCl₂, 0.08M Tris pH 8.5, 16% w/v PEG3350. Crystals were transferred to a cryo-protectant consisting of 25% ethylene glycol and 75% well solution before flash-cooling in liquid nitrogen.

Data Collection: Resolution: 1.9Å; X-ray source: SLS beam X10SA.