Human FK506-binding protein 6

Target ID:

FKBP06

Aliases:

FKBP36MGC87179PPIase

Deposition Date:

Wednesday 31st October 2007

Families:

Isomerases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3B7X

Authors:

Walker, J.R., Davis, T., Paramanathan, R., Butler-Cole, C., Weigelt, J., Sundstrom, M., Arrowsmith, C.H., Edwards, A.M., Bochkarev, A., Dhe-Paganon, S.

Site:

Toronto

ICM Datapack:

ICM Datapack

3B7X

tabs

Structure Details

Homologous recombination is a necessary part of the production of sperm and egg. It involves the exchange of DNA fragments between equivalent chromosomes and its starts with the unification of chromosomal pairs (synapses) followed by cutting and joining crosswise at particular points (chiasmata). This process enhances genetic diversity but can also lead to deletions and duplications that may cause disease; however, it is likely the basis of evolution. A heterotrimeric protein assembly (SYCP1, 2, and 3) referred as the synaptonemal complex has been identified as the enzyme that catalyzes homologous recombination. A fourth component, FKBP6, which is linked to Williams syndrome, has recently been discovered as an important element of the enzyme (Meng et al., 1998; Crackower et al., 2003). Its molecular function during homologous recombination as well as its role in Williams syndrome is unknown.

We have determined the high resolution crystal structure of the catalytic domain of human fkbp6, revealing the canonical fold with a beta-bulge. Unique features include aliphatic side chains in the active site pocket, an expanded beta2-beta3 loop, and an amino-terminal alpha-helix that wraps around the core domain with its very N-terminus interacting with the expanded beta2-beta3 loop. Higher order structural studies will address its role in the synaptonemal complex.

Materials and Methods

FKBP6

PDB:3B7X

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_003593
Entry Clone Source:fkbp06.BC036817.MGC.AT57B11.pCMVSPORT6
SGC Clone Accession:fkbp06.012.144.108B02 (SDC108B02)
Tag:MHHHHHHSSGRENLYFQG
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgEGDDAPGQSLYERLSQRMLDISGDRGVLKDVIREGAGDLVAPDASVLVKYSGYLEHMDRPFDSNYFRKTPRLMKLGEDITLWGMELGLLSMRRGELARFLFKPNYAYGTLGCPPLIPPNTTVLFEIELLDFLD
Vector:p28a-mhl

Growth

Medium:
Antibiotics:
Procedure:A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 degC.

Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37 degC. The temperature of the media was reduced to 15oC one hour prior to induction and induced at OD600 = 6 with 100 uM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC, and cell pellets collected by centrifugation and frozen at -80 degC.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of HisLink Protein Purification Resin (Promega V8821) per 40 mL lysate. The mixture was incubated with mixing for at least 20 minutes at 4oC. The lysate was spun at 500 xg for 5 minutes, and the supernatant was decanted. The remaining resin was washed with 45 mL of cold wash buffer, allowing 5 minutes to settle between washes, until the supernatant was clear (usually 3-5 washes). The washed resin was transferred to a gravity column and further washed with 1 column volumes (approx. 5 mL) of wash buffer at approximately 3 mL/min. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10 mL) of elution buffer.

Gel filtration chromatography: An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column at a flow rate of 2 mL/min. The eluate sample from the IMAC step (approx. 10 mL) was loaded onto the column at 2 mL/min. Peak fractions are inspected and pooled.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture are thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration of 10 mg/mL.
Ligand
MassSpec:Mass-spectroscopy by LCMS showed pure product of correct molecular weight
Crystallization:Crystals of the FKBP36 isomerase domain were grown at 298 K using the sitting drop method by mixing equal volumes of 1.6M MgSO₄, 0.1M MES pH6.5, and 10 mg/mL protein. The crystals were cryoprotected by transferring the crystals to a drop containing mother liquor to which glycerol was added to a final concentration of 20%.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Crackower,M.A., Kolas,N.K., Noguchi,J., Sarao,R., Kikuchi,K., Kaneko,H., Kobayashi,E., Kawai,Y., Kozieradzki,I., Landers,R., Mo,R., Hui,C.C., Nieves,E., Cohen,P.E., Osborne,L.R., Wada,T., Kunieda,T., Moens,P.B., and Penninger,J.M. (2003). Essential role of Fkbp6 in male fertility and homologous chromosome pairing in meiosis. Science 300, 1291-1295.
Meng,X., Lu,X., Morris,C.A., and Keating,M.T. (1998). A novel human gene FKBP6 is deleted in Williams syndrome. Genomics 52, 130-137.