Human complement component 1, q subcomponent binding protein

Target ID:

C1QBP

Deposition Date:

Wednesday 27th April 2011

Families:

Miscellaneous

Related Diseases:

CancerMetabolism

Structure type:

apo

Download Coordinates:

3RPX

Authors:

Tempel W, Tong Y, Crombet L, Shen Y, Guan X, Nedyalkova L, Walker JR, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Park H

Site:

Toronto

3RPX

tabs

Structure Details

C1QBP, complement component 1, Q subunit binding protein, is a versatile protein involved in many biological functions. C1QBP inhibits complement 1 system by binding with the Q subunit, regulates in mRNA processing by inhibiting SF2 splicing factor, recognizes multiple glycosylated proteins through interaction with hyaluronan, and may also serve as a divalent cation storage protein. Recently C1QBP was found to critically regulate tumor metabolism by protecting oxidative phosphorylation and the expression level of C1QBP correlates with prognosis of prostate cancer. It was demonstrated to be a potential tumor therapy target.

An N-terminal helix truncated version of C1QBP was expressed in E. coli and purified using IMAC affinity chromatography. The crystal structure of the construct was solved at 2.65 Å resolution. C1QBP forms a ring-like homotrimer in the asymmetric unit. Compared to previous solved, longer version of C1QBP, (PDB:1P32), our crystal was grown at a condition closer to physiological pH (=7.5) and salt condition (0.2 M NaCl). Our structure suggests the N-terminal helix is not critical for homotrimerization as proposed previously [1], and trimerization is an intrinsic property of the protein.

Materials and Methods

C1QBP

PDB:3RPX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC000435
Entry Clone Source:MGC:AU1-D11
SGC Clone Accession:HPC09Q-F01
Tag:C-terminal His6-tag
Host:BL21(DE3)-V2R-pRARE2

Construct

Prelude:Construct ID C1QBP:P3-Q282:CH
The construct was designed to encode residues P3-Q282. However, DNA sequence encoding residues from a.a. 18 to 88 was skipped in the PCR products, probably due to strange DNA secondary structure and the resulting plasmid encodes a truncated protein of E89-Q282 and the reading frame was not right.
The measured mass of the purified protein was 22119.6, the closest construct will be I96-Q282 (m.w. expected 22102.23).
Sequence:IQKHKTLPKMSGGWELELNGTEAKLVRKVAGEKITVTFNINNSIPPTFDGEEEPSQGQKVEEQEPELTSTPNFVVEVIKNDDGKKALVLDCHYPEDEVGQEDEAESDIFSIREVSFQSTGESEWKDTNYTLNTDSLDWALYDHLMDFLADRGVDNTFADELVELSTALEHQEYITFLEDLKSFVKSQahhhhhhThe exact N-terminus is not clear
Vector:pNIC-CH

Growth

Medium:Terrific Broth medium in the presence of 50 mg/mL kanamycin and 25 mg/mL chloramphenicol
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating 100 mL of overnight culture grown in Luria-Bertani medium into a 2 L of growth medium at 37 °C. When OD₆₀₀ reached ~3.0, the temperature of the medium was lowered to 15 °C and the culture was induced with 1 mM IPTG. The cells were allowed to grow overnight before harvested and flash frozen in liquid nitrogen and stored at -80 °C.

Purification

Procedure
The lysate was centrifuged at 16,000 rpm for 60 minutes and the supernatant were mixed with 6 mL 50% flurry of Ni-NTA beads and incubated at 4 degrees Celsius on rotary shaker for 60 minutes. The mixture was then centrifuged at 2300 rpm for 5 min and the supernatant discarded. The beads were then washed with 100 mL binding buffer containing 5 mM Imidazole followed by 50 mL washing buffer. Bound proteins were eluted using 15 mL elution buffer. The flow-through was collected and further purifed by a Superdex-75 26/60 gel filtraton column pre-equilibrated with gel filtration buffer. Fractions containing the target protein were pooled and concentrated using Amicon Ultra-15 centrifugal filter (mwco 10 kDa). The purity of the preparation is tested by SDS-PAGE to be greater than 90%.

Extraction

Procedure
Frozen cells from 4L TB culture were thawed and resuspended in 500 mL extraction buffer with freshly added 0.5% CHAPS, and supplemented with 1.8 mL protease inhibitor cocktail (SIGMA Catalog # P8849), and 10 uL benzonase (Sigma Catalog # E1014, 250U/uL), 1mM PMSF/Benzamidine, and lysed using sonication for 8 min at 120 W, 10 sec on/10 sec off duty cycle.
Concentration:24 mg/mL
Ligand
MassSpec:Native protein measured 22119.6
Crystallization:Crystal was initially obtained from SGC-I screen condition H7 (also available from SGC:A8,D6).
Crystal used for structure refinement was grown in 25% PEG 1500, 0.2 M NaCl, 0.1 M HEPES buffer pH 7.5, 5% Glycol sitting drop setup, using 0.3 uL protein + 0.3 uL well solution against 100 uL reservoir buffer at room temperature.

Crystals grow to a mountable size within a few days.
Cryo used paratone-N
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Jiang J, Zhang Y, Krainer AR, Xu RM. Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc. Natl Acad. Sci. U.S.A 1999, 96:3572-3577.