Human 3-hydroxyisobutyryl-Coenzyme A hydrolase + inhibitor

Target ID:

HIBCHA

Aliases:

HIBCHHIBYL-COA-H

Deposition Date:

Wednesday 19th December 2007

Families:

Miscellaneous

Related Diseases:

MetabolismNeurobiology

Structure type:

protein-ligand

Download Coordinates:

3BPT

Authors:

Pilka, E.S., Phillips, C., King, O., Guo, K., von Delft, F., Pike, A.C.W., Arrowsmith, C.H., Weigelt, J., Edwards, A., Oppermann, U.

Site:

Oxford

3BPT

tabs

Structure Details

The catabolic pathway of the amino acid valine proceeds through an intermediate hydrolysis of 3-hydroxyisobutyryl CoA to the free organic acid, catalyzed by the enzyme 3-hydroxyisobutyryl-CoA hydrolase (HIBCH). The hydrolysis of a CoA intermediate in valine metabolism is in marked contrast to the degradation of the other branched chain amino acids, in which the intermediates distal to the 2-keto acid products are all thioesters.

The human HIBCH gene is localized to chromosome 2q32.2. The enzyme is predicted to be localized to mitochondria, and shows a wide tissue distribution, with high expression in liver, kidney, heart and lung.

Although very rare, mutations in the human HIBCH gene have been identified and linked to severe developmental abnormalities. Although not fully investigated at present, HIBCH deficiency possibly leads to accumulation of a reactive methacrylyl-CoA intermediate. The identified patients suffer from delayed development of motor skills, hypotonia, progressive degeneration of neurological functions, and abnormalities in brain development, ultimately leading to early death.

Materials and Methods

HIBCH

PDB:3BPT

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:5266813
Entry Clone Source:MGC
SGC Clone Accession:
Tag:C-terminal, TEV cleavable hexahistidine tag. Tag sequence: aenlyfq(*)shhhhhhdykddddk
Host:E. coli B834 (DE3) for SeMet labelling

Construct

Prelude:
Sequence:

MTDAAEEVLLGKKGCTGVITLNRPKFLNALTLNMIRQIYPQLKKWEQDPETFLIIIKGAGGKAFCAGGDIRVISEAEKAKQKIAPVFFREEYMLNNAVGSCQKPYVALIHGITMGGGVGLSVHGQFRVATEKCLFAMPETAIGLFPDVGGGYFLPRLQGKLGYFLALTGFRLKGRDVYRAGIATHFVDSEKLAMLEEDLLALKSPSKENIASVLENYHTESKIDRDKSFILEEHMDKINSCFSANTVEEIIENLQQDGSSFALEQLKVINKMSPTSLKITLRQLMEGSSKTLQEVLTMEYRLSQACMRGHDFHEGVRAVLIDKDQSPKWKPADLKEVTEEDLNNHFKSLGSSDLKFaenlyfq

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

Vector:pNIC-CTHF

Growth

Medium:
Antibiotics:
Procedure:A single colony of B834 (DE3) was added to 200ml of LB with 50µg/ml kanamycin and grown at 30°C overnight. Harvested cells were washed twice with PBS and resuspended in MD medium. 10ml were inoculated into 1L of MD medium with 50µg/ml kanamycin and 40mg of Selenomethionine powder (8L in total) Cultures were grown at 37°C. At the OD of 1 the temperature was lowered to 25°C and an hour later 0.5mM IPTG was added. The next day cells were harvested by centrifugation at 4000 rpm for 10min and stored at -20°C.

Purification

Procedure

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

Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 40mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole, 0.5mM TCEP.

GF buffer: 50mM HEPES pH 7.5, 150mM NaCl, 0.5mM TCEP

Procedure: The cell extract was loaded on an AKTA Express system. The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage. A single resulting peak was characterised by mass spectrometry.

TEV cleavage: The His-tag was cleaved with 1mg TEV per 40mg target protein at 4°C overnight.

Extraction

Procedure
Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml). The thawed cells were resuspended in the lysis buffer and broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45min at 15.000rpm.
Concentration:Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 30 kDa cutoff, the sample was concentrated to 65mg/ml. Concentrations were determined from the absorbance at 280 nm using NanoDrop. Electron density has shown the N-terminal SeMet to be present.
Ligand
quercetinMassSpec:The calculated mass of the construct was 41026. The exact mass was confirmed by mass spectrometry, together with a second peak arising from the lack of the N-terminal SeMet (40848).
Crystallization:Crystals were grown by vapor diffusion at 20°C in 320nl sitting drops. Quercetin was added to a final concentration of 5mM prior to crystallisation. The drops were prepared by mixing 100nl of protein solution and 200nl of precipitant consisting of 0.1M citrate pH 5.5 and 20% PEG 3350. Then the drops were seeded with 20ul of suspended micro-seeds created from the native crystals. Crystals were transferred to a cryo-protectant consisting of the well solution and 20% PEG 400 before flash-cooling in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 1.7Å; X-ray source: SLS beam X10SA.
Data Processing: