Human kinesin light chain 1, tetratricopeptide repeat domain

Target ID:

KNS2

Deposition Date:

Wednesday 9th June 2010

Families:

Miscellaneous

Related Diseases:

CancerNeurobiology

Structure type:

apo

Download Coordinates:

3NF1

Authors:

Tong Y, Tempel W, Shen L, Shen Y, Nedyalkova L, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Bochkarev A, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3NF1

tabs

Structure Details

Conventional kinesins are heterotetramer composed of two light chains and two heavy chains. The light chains interact with the tail domain of heavy chains and together they form the cargo binding region.

TPR (tetratricopeptide repeat) motif is a protein-protein interaction module containing 34 amino acids loosely based around the consensus residues -W-LG-Y-A-F-A-P-. Kinesin light chain 1, KLC1 contains 6 TPR motifs and is known to bind amyloid precursor protein (APP) and responsible for the transport of APP vesicles (Kamal et al., 2000). Misregulation of the APP vesicles transport is one possible cause of the Alzheimer's disease (Stokin et al., 2005).

We solved the crystal structure of the KLC1 TPR domain at 2.8 A resolution. Each TPR repeat forms a helix-turn-helix architecture. There is one extra helix inserted between TPR5 and TPR6.

Materials and Methods

KLC1

PDB:3NF1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AU40-H2
Entry Clone Source:MGC library
SGC Clone Accession:HPC09H-C05
Tag:mhhhhhhssgrenlyfqg
Host:BL21-V2R-pRARE2

Construct

Prelude:KNS2:G205-G497
Tag not removed
Sequence:mhhhhhhssgrenlyfqgGGYEIPARLRTLHNLVIQYASQGRYEVAVPLCKQALEDLEKTSGHDHPDVATMLNILALVYRDQNKYKDAANLLNDALAIREKTLGKDHPAVAATLNNLAVLYGKRGKYKEAEPLCKRALEIREKVLGKDHPDVAKQLNNLALLCQNQGKYEEVEYYYQRALEIYQTKLGPDDPNVAKTKNNLASCYLKQGKFKQAETLYKEILTRAHEREFGSVDDENKPIWMHAEEREECKGKQKDGTSFGEYGGWYKACKVDSPTVTTTLKNLGALYRRQGKFEAAETLEEAAMRSRKQG
Vector:pET28-MHL

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 50 mL of overnight culture grown in Luria-Bertani medium into a 2L of Terrific Broth medium in the presence of 50 mg/mL kanamycin and 25 mg/mL chloramphenicol 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 15,000 rpm for 60 minutes and the supernatants were mixed with 4 mL 50% flurry of Ni-NTA beads and incubated at 4 degree on rotary shaker for one hour. The mixture was then centrifuged at 2500 rpm for 5 min and the supernant discarded. The beads were then washed with washing buffer, and finally the elution buffer. The flow-through was collected and further purifed by a Superdex-75 gel filtraton column pre-equilibrated with gel filtration buffer. Fractions containing the protein were collected and concentrated with Amicon Ultra-15 centrifugal filter. The purity of the preparation is tested by SDS-PAGE to be greater than 95%.

Extraction

Procedure
Frozen cells from 4L TB culture were thawed and resuspended in 150 mL extraction buffer with freshly added 0.5% CHAPS, and supplemented with protease inhibitor cocktail (SIGMA Catalog # P8849), and 3 uL benzonase (Sigma Catalog # E1014, 250U/uL), and lysed using sonication.
Concentration:20.5 mg/mL
Ligand
MassSpec:Protein expected 35379; measured 35380.2
Crystallization:Crystallization was setup using sitting drops with Red Wings and SGC-I screens in 0.25 uL protein solution plus 0.4 uL well solution ratio. Diffracting crystals were found from initial screen plate for Red Winds E2. Crystal used for structure determination was grown in 2.0M Ammonium Formate, 0.1M HEPES buffer at pH 7.5, 5mM TCEP. Paratone was used as cryoprotectant. Crystals grow to a mountable size within one week.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Kamal, A., Stokin, G. B., Yang, Z., Xia, C. H., & Goldstein, L. S. (2000). Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I. Neuron, 28(2), 449-459.

Stokin, G. B., Lillo, C., Falzone, T. L., Brusch, R. G., Rockenstein, E., Mount, S. L., Raman, R., Davies, P., Masliah, E., Williams, D. S., & Goldstein, L. S. (2005). Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science, 307(5713), 1282-1288.