Human kinesin family member C1

Target ID:

KIFC1

Aliases:

HSETKNSL2MGC1202MGC149736MGC149737MKLP2RAB6-KIFL

Deposition Date:

Wednesday 26th September 2007

Families:

Miscellaneous

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

2REP

Authors:

Zhu H, Tempel W, He H, Shen Y, Wang J, Brothers G, Landry R, Arrowsmith CH, Edwards AM, Sundstrom M, Weigelt J, Bochkarev A, Park HW

Site:

Toronto

ICM Datapack:

ICM Datapack

2REP

tabs

Structure Details

Kinesin superfamily proteins (KIFs) have been shown to hydrolyzing ATP for energy to transport organelles, protein complexes and mRNAs to specific destinations along microtubules. KIFs also participate in chromosomal and spindle movements during mitosis and meiosis. An approximately 360-residue globular domain is the hallmark of KIFs. This well-conserved domain contains both a catalytic pocket for the hydrolysis of ATP and the binding sites for microtubules. It is often referred to as the 'catalytic core' [1].

Structure Features

KIFC1 belongs to the kinesin 14A family. The motor domain locates at the C-terminus of the kinesin heavy chain. KIFC1 moves toward the minus-end of microtubules and is a mitotic motor functioning in nuclear fusion in yeast [2] or multiple functions in higher organisms [3]. Although not proven yet, the mitotic kinesins are emerged as druggable targets. Here, we present the structure of the motor domain of KIFC1 in complex with Mg2+ and ADP. The motor domain of KIFC1 adapts a similar folding topology to other kinesin motor domains: the three-layer (αβα) sandwich architecture. Three α-helices are at each side of an eight β-stranded sheet (e.g., the rmsd value of 1.9Å to the Eg5 motor domain). Similar to the known ATP-binding proteins, four conserved motifs (N1-N4) are found in the KIFC1 motor domain [4]. They are N1 (410GQTGSGKT417), N2 (527ARRSSR215), N3 (565DLAGSE594) and N4 (316RVRP319).

Materials and Methods

KIFC1

PDB:2REP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:XP_371813
Entry Clone Source:MGC
SGC Clone Accession:HPC042-B8
Tag:N-terminal hexahistidine tag
Host:Hi-Five insect cells

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsLKGNIRVFCRVRPVLPGEPTPPPGLLLFPSGPGGPSDPPTRLSLSRSDERRGTLSGAPAPPPRHDFSFDRVFPPGSGQDEVFEEIAMLVQSALDGYPVCIFAYGQTGSGKTFTMEGGPGGDPQLEGLIPRALRHLFSVAQELSGQGWTYSFVASYVEIYNETVRDLLATGTRKGQGGECEIRRAGPGSEELTVTNARYVPVSCEKEVDALLHLARQNRAVARTAQNERSSRSHSVFQLQISGEHSSRGLQCGAPLSLVDLAGSERLDPGLALGPGERERLRETQAINSSLSTLGLVIMALSNKESHVPYRNSKLTYLLQNSLGGSAKMLMFVNISPLEENVSESLNSLRFASKVNQC
Vector:pFBOH-LIC

Growth

Medium:
Antibiotics:
Procedure:Transposition: 2 µl of the construct was added and mixed to 30 µl of DH10Bac competent cells in a sterile 96-well microtitre plate on ice. The plate was left on ice for a further 30 minutes. The heat-shock procedure was done by transferring the plate to a 42°C water bath for 60 seconds and then returning it to ice for a further 2 minutes. 600 µl of SOC medium (pre-warmed to 37 °C) was added to the well and the plate incubated at 37°C for 5 hours. The 2 µl culture mixed with pre-warmed 100 µl SOC, and plated out onto LB agar in a 5.5 cm Petri dish contains Gentamicin (7 µg/ml), Kanamycin (50 µg/ml) and Tetracycline (10 µg/ml). The plates were incubated at 37°C for 48 hours.

Bacmid preparation: One white colony was picked into 3 ml of LB media, with Gentamicin (7 µg/ml), Kanamycin (50 µg/ml) and Tetracycline (10 µg/ml), in a 24-well block (Qiagen, Cat. 19583) and placed in a shaker (250 rpm) for 18 hours at 37°C. Bacmids were purified with Montage® kit (Millipore Cat. LSKB09604).

Generation of P1 recombinant Baculovirus: In a Napflow® Class II type A/B3 biosafty cabinet, 50 µl HyQ® SFX-insect serum medium (Hyclone, Cat. SH30278.02) was added into 6 µg bacmid and cellfectin (Invitrogen Cat. 10362-010). Then bacmid and cellfactin in the medium were mixed and incubated at room temperature for 45 minutes. 1 ml SF9 cells (2 x 10⁵ cells/ml) in HyQ® SFX-insect serum medium was added into the mixture in a 24 well plate (Falcon Cat. 353047). After cells sat at the bottom of the plate, remove supernatant, and 280 µl HyQ® SFX-insect serum medium was added to the plate, then the plate was incubated at 27 ºC for 5 hours. In the plate, the supernatant of the mixture was replaced with 0.7 ml GraceÂs insect medium (Invitrogen Cat. 11595-030) contained 10% FBS (Invitrogen Cat.12483-020) and 1% antibiotics (100 µg/ml penicillin, 100 µg/ml streptomycin). Then the plate was incubated in 27 ºC for 72 hours. The supernatant was collected.

Generation of P2 recombinant Baculovirus: In a 6 well plate (Falcon Cat. 353047), SF9 cells (1 x 10⁶ cells / ml) in 1.5 ml HyQ® SFX-insect serum medium were infected with 80 µl P1 viruses in 27 °C. The culture was incubated in 27 °C for 48 ~ 72 hours. Supernatant was collected after incubation.

Generation of P3 recombinant Baculovirus: In a 500 ml flask, high-five cells were added into HyQ® SFX-insect serum medium to reach the desity of 2 x 10⁶ cells / ml. 0.2 ml of P2 recombinant Baculovirus was added into the culture. The flask was shaken in 27 °C, 130 rpm for 48 hours. Supernatant was collected.

Protein production: 10 ml P3 recombinant Baculovirus cells were added into 1 L HyQ® SFX-insect serum medium contained High-Five cells (2 x 10⁶ cells / ml) and Gentamicin (10 µg / ml). The culture was put on a shaker with 100 rpm, at 27 °C for 48 hours. Cells were harvested with centrifuge (4000 rpm, 20 minutes). Harvested cells were washed with cold PBS buffer, then flash frozen in liquid nitrogen and stored at -80 °C.

Purification

Procedure
Column 1: Ni-NTA beads
Column 2: Size exclusion chromatography (Superdex 75 26/60)

1 ml of Ni-NTA suspension solution was added into 40 ml cell lysis supernatant solution. The mixture was shaken for 1 hour at 4 ºC. Beads were collected with centrifuge at 2500 rpm, 5 minutes. Beads were washed with 25 ml washing buffer, then collected with centrifuge. Protein was eluted with 15 ml elution buffer.

The fractions eluted of the Ni-affinity chromatography applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 1.0 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

Extraction

Procedure
Cell breakage: 1 passes through the Emulsiflex C5 high pressure homogeniser. Centrifuge for 60 mins at 16000 rpm and 4°C to remove cell debris. Discard pellet.
Concentration:Centricon with a 10 kDa cut off in SEC-buffer. The final concentration is 11.2 mg/ml.
Ligand
MassSpec:measured: 40289.3 Da.
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 16 mg/ml containing 5 molar fold of ADP and MgCl2. 0.5 µl of the concentrated protein mixed with 0.5 µl of a well solution containing 3.5 M NaCl, 0.1 M Bis-Tris Propane pH 7.50. Crystals appeared after a day at 18°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using mother liquor containing 20% glycerol, and flash frozen in liquid nitrogen. Diffraction data were collected APS 19-ID to 2.6 Å.
Data Processing:

References

1. Miki, H., Okada, Y., Hirokawa, N., (2005) Analysis of the kinesin superfamily insights into structure and function Trends in cell. Biolo., 15, 467-476.

2. Meluh, P.B. and Rose, M.D. (1990) KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60, 1029-1041.

3. Kuriyama, R. et al. (1995) Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells. J. Cell Biol. 129, 1049-1059.

4. Zou, Y. et al. (2002) KRP3A and KRP3B: candidate motors in spermatid maturation in the seminiferous epithelium. Biol. Reprod. 66, 843-855