Human kinesin family member 9

Target ID:

KIF9

Aliases:

MGC104186

Deposition Date:

Wednesday 1st November 2006

Families:

Miscellaneous

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

Superceded by 3NWN

Authors:

H.ZHU,W.TEMPEL,H.HE,Y.SHEN,J.WANG,G.BROTHERS,R.LANDRY,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,H.PARK,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2NR8

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 while. 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. Hence, it is often referred to as the catalytic core[1].

The designation 9 was taken from the family member KIF9. This is a novel family identified by the familyspecific neck adjacent to and downstream of the catalytic core. The amino acid sequence in the neck is conserved within this family. Catalytic core sequences also loosely stablish this family. Contrary to some reports [2, 3], The precise function of family members is still uncertain; however, the absence of this family in higher plants suggests a function in cilia and flagella [4], as in the case of Kinesin-2 [1].

The structure presented is the motor domain of KIF 9 in complex with Mg2+ and ADP. Superimposing our structure (see iSee data pack) with the motor domain (PDB: 1GOJ in blue) of the fast fungal kinesin from Neurospora crassa shows that the overall folding KIF 9 motor domain of is very similar to conventional N-terminal motor domain except from residues H36 to residues D52 form an extra coil and helix. It has three layers (αβα) sandwich architecture. Three α-helixes are at each side of eight β-sheets. As found in other ATP-binding proteins, four conserved motifs (N1-N4) were found in the structure [5]. The phosphate-binding loop or N1 (93GQTGAGKT100), Sw1 or N2 (210NKNSSR215), Sw2 or N3 (244DLAGSE249) and L1 or N4 (12RVKP15), which interacts with the base.

Materials and Methods

KIF9

PDB:2NR8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:34193691
Entry Clone Source:
SGC Clone Accession:
Tag:
Host:Hi-Five insect cells.

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsGTRKKVHAFVRVKPTDDFAHEMIRYGDDKRSIDIHLKKDIRRGVVNNQQTDWSFKLDGVLHDASQDLVYETVAKDVVSQALDGYNGTIMCYGQTGAGKTYTMMGATENYKHRGILPRALQQVFRMIEERPTHAITVRVSYLEIYNESLFDLLSTLPYVGPSVTPMTIVENPQGVFIKGLSVHLTSQEEDAFSLLFEGETNRIIASHTMNKNSSRSHCIFTIYLEAHSRTLSEEKYITSKINLVDLAGSERLGKSGSEGQVLKEATYINKSLSFLEQAIIALGDQKRDHIPFRQCKLTHALKDSLGGNCNMVLVTNIYGEAAQLEETLSSLRFASRMKLV
Vector:pFBOH-LIC

Growth

Medium:
Antibiotics:
Procedure: Transposition: 2 microL 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 degC water bath for 60 seconds and then returning it to ice for a further 2 minutes. 600 µl of SOC medium (pre-warmed to 37degC) 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 37degC 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 37degC. Bacmids were purified with Montage(R) kit (Millipore Cat. LSKB09604).
Generation of P1 recombinant Baculovirus: In a Napflow(R) Class II type A/B3 biosafty cabinet, 50 µl HyQ(R) 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 °C for 5 hours. In the plate, the supernatant of the mixture was replaced with 0.7 ml Grace?s 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 °C 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 degC.

Purification

Procedure
Column 1: Ni-NTA beads

Column 2: Size exclusion chromatography (Superdex 200 26/60)
The fractions eluted of the Ni-affinity chromatography applied to a Superdex S200 column equilibrated in SEC buffer at a flow rate of 2.0 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.Concentration:Centricon with a 10kDa cut off in SEC-buffer. Protein concentrations were measured using Bradford assay with purity >95% based on SDS-PAGE analysis.

Extraction

Procedure
50 mM Hepes, pH 8.0, 0.5 M NaCl, 5% glycerol (v/v), and 5 mM imidazole, 1 mM TCEP. Six EDTA-free Complete® protease inhibitor tablets in 100 ml of extraction buffer, 1.2 ml of 0.05 M PMSF, 1.2 ml 0.1 M benzamidine and 3750 units of benzonase were added in to extraction buffer. 10g pellets / 100 ml extraction buffer. 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:
Ligand
MassSpec:
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 1.5 M (NH4)2SO4, 0.1 M Tris, pH 8.50. Crystals appeared after a day at 18 °C.
NMR Spectroscopy:
Data Collection:Resolution: 2.0Å, X-ray source: Crystals were cryo-protected using 50% mineral oil and 50% parotone, and flash frozen in liquid nitrogen. Diffraction data were collected at the home source to 2.0Å.
Data Processing:

References

Miki, H., Okada, Y., Hirokawa, N., (2005) Analysis of the kinesin superfamily insights into structure and function Trends in cell. Biolo., 15, 467-476.
Zou, Y. et al. (2002) KRP3A and KRP3B: candidate motors in spermatid maturation in the seminiferous epithelium. Biol. Reprod. 66, 843-855
Piddini, E. et al. (2001) The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9. EMBO J. 20, 4076-4087
Bernstein,M. et al. (1994) A new kinesin-like protein (Klp1) localized to a singlemicrotubule of the Chlamydomonas flagellum. J. Cell Biol. 125, 1313?1326
Marx, A., Mueller, J., Mandelkow, E., (2005) The structure of microtubule motor proteins Adv Protein Chem., 71, 299-341.