Human propionyl-CoA carboxylase (N-term, biotin acceptor domain)

Target ID:

PCCAA

Aliases:

PCCA

Deposition Date:

Saturday 30th August 2008

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

2JKU

Authors:

TBA

Site:

Oxford

2JKU

tabs

Structure Details

PCCA is a biotin-attachment domain from propionyl-CoA carboxylase (PCC), a mitochondrial enzyme involved in the catabolism of branched chain amino acids and odd-chain fatty acids, by catalyzing carboxylation of propionyl-CoA, producing D-methylmalonyl-CoA. PCC consists of two non-identical subunits (αβ) to form an α6β6 oligomer. The biotin-attachment domain is part of the α subunit at the C-terminus. The α subunit also contains a biotin carboxylase domain at the N-terminus. This domain contains ATP and bicarbonate binding sites, which acts by transferring a carboxy group onto biotin, forming carboxybiotin in an ATP-dependent reaction. The second half of the reaction, involving the β subunit, transfers the carboxy group from biotin onto propionyl-CoA. Through the activity of holocarboxylase synthetase, biotin is attached to a lysine within the consensus sequence Ala-Met-Lys-Met in the biotin-attachment domain, which is conserved in all biotin-dependent carboxylases,.

Mutations in the PCC gene lead to propionic academia, a rare neonatal disorder characterized by metabolic ketoacidosis, lactic acidosis, vomiting, lethargy and hypotonia. A range of symptoms may be presented, and in most patients there is associated developmental delay and early death, though some patients do well on a protein restricted diet. There are 24 known mutations on the α subunit, a number of which are within the biotin-binding domain, and 29 known mutations on the β subunit.

We have determined the structure of the N-terminal region of the biotin-attachment domain.

Materials and Methods

Entry Clone Source: n/a

Entry Clone Accession: GI:34189907

SGC Construct ID: PCCAA-c002

GenBank GI number: gi|65506442

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

Tags and additions: N-terminal, TEV cleavable hexahistidine tag. Tag sequence: mhhhhhhssgvdlgtenlyfq(*)sm

Host: BL21(DE3)-R3-pRARE2

Sequence (tag sequence in lower case) :
mhhhhhhssgvdlgtenlyfqs*mTSSVL
RSPMPGVVVAVSVKPGDAVAEGQEICVIE
AMKMQNSMTAGKTGTVKSVHCQAGDTVGE
GDLLVELE

Expression: Rosetta cells were grown in 1x TB medium supplemented with 50 µg/ml kanamycin at 37°C. Cells were induced at an optical density of 0.9 – 1.0 with 0.1 M IPTG at 18°C. 20 hours post-induction the cultures were collected and centrifuged for 30min at 15000rpm. The pellet was resuspended in lysis buffer (50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml)) and frozen at -20°C until purification.

Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml). The thawed cellular lysates were supplemented with benzonase (Novagen) (25u/10ml of lysate). Cells were lysed with sonication (20 minutes, 5 sec pulse on, 5 sec cooling), followed by centrifugation for 60 min at 15000rpm at 4°C.

Column 1: Ni-affinity, Ni-Sepharose - (GE Healthcare) purification in batch

Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole.

Procedure: The cell extract was incubated with Ni-Sepharose (5 mL of resin/lysate from 2 l of culture) during 1h at 4°C with gentle rotation. The resin was centrifuged at 1000g for 5min at 4°C, supernatant was removed and the resin was loaded on the gravity column. Resin was washed 4x with 50ml of washing buffer. Protein was eluted in 10 elution fractions (2 ml each). Protein fractions were analysed by SDS-PAGE. Target protein containing fractions were concentrated and buffer was exchanged into lysis buffer using Amicon Ultra-15 concentrators with 3kDa cut-off. Protein was digested overnight with TEV (60 µg/mg target protein). TEV and His-tag were removed by mixing with 50 µL Ni-resin for 1 hour, and washing resin 3x 2mL with lysis buffer.

Concentration and buffer exchange: Using PD10 desalting columns, the buffer was exchanged into 50mM HEPES, 100mM NaCl, pH 7.5. The sample was concentrated to 21mg/ml. Concentrations were determined from the absorbance at 280 nm using a NanoDrop spectrophotometer.

Mass spec characterization: The experimental mass coincides with th theoretical mass (7463Da).

Crystallization: Crystals were grown by vapor diffusion at 20°C in 150nl sitting drops. The drops were prepared by mixing 100nl of protein solution and 50nl of precipitant consisting of 20% PEG3350, 0.2M sodium formate. Crystals were flash-cooled in liquid nitrogen with 25 % ethylene glycerol as cryoprotectant.

Data Collection: Resolution: 1.5Å. X-ray source: Swiss Light source (SLS), beamline X-10.