Human fragile X mental retardation, autosomal homolog 2, Tudor domains

Target ID:

FXR2

Deposition Date:

Wednesday 29th April 2009

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3H8Z

Authors:

Amaya MF, Adams-Cioaba MA, Guo Y, MacKenzie F, Kozieradzki I, Edwards AM, Arrowsmith CH, Bochkarev A, Min J.

Site:

Toronto

ICM Datapack:

ICM Datapack

3H8Z

tabs

Structure Details

Recognition of protein methylation has been functionally ascribed to members of several protein families including the PHD domain (Taverna et al. 2007), the "royal family (Maurer-Stroh et al., 2003)," and the WD40 repeat protein WDR5 (Fischle et al. 2003;Flanagan et al. 2005;Jacobs et al. 2002;Min et al. 2003;Nielsen et al. 2002;Wysocka et al. 2005). The "royal family" of proteins includes the Tudor, plant Agenet, chromo, PWWP and MBT domains, many of which have been found in association with chromatin (Maurer-Stroh et al. 2003). The recognition of post-translational methylation has traditionally been studied in the context of histone regulation (Shi, Y., and Whetstine, J. R. 2007). More recently, however, methyation of both lysine and arginine have also been shown to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. 2008). Through a rapidly expanding body of research, Tudor domains have been identified within numerous proteins involved in RNA metabolism and other cellular signaling pathways, in addition to their involvement in the recognition of histone methylation sites.

The fragile X mental retardation -related protein 2 (FXR2) is a 74kDa protein comprising N-terminal tandem Tudor Domains, a helix-loop-helix motif, tandem KH-type domains and an RG-box. The protein is homologous to fragile X mental retardation protein, the loss of which underlies fragile X syndrome. While the tissue and cellular expression patterns of these proteins are highly similar, studies of knock-out mice suggest that the members of this protein family are important in regulating transcription/translation of distinct populations of genes. The tandem Tudor domains of FXR2 retain canonical β barrel folds that are arranged with high similarity to those of UHFR1 (PDB 3DB4 ).

Materials and Methods

FXR2

PDB:3H8Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession: BC067272
Entry Clone Source:MGC AT94-B9,
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgrenlyfq*g.
Host:E. coli BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgLPVEVRGSNGAFYKGFVKDVHEDSVTIFFENNWQSERQIPFGDVRLPPPADYNKEITEGDEVEVYSRANEQEPCGWWLARVRMMKGDFYVIEYAACDATYNEIVTLERLRPVNPNPLATKGSF
Vector:pET28-MHL

Growth

Medium:M9 SeMET groth media (Medicilon Inc).
Antibiotics:
Procedure:A fresh transformation was used to inoculate 20 mL LB media containing 50 µg/mL kanamycin and 30 µg/mL chloramphenicol . The culture was grown overnight at 37°C with shaking. The next day this starter culture was used to innoculate 2L of M9 SeMET growth medium. The culture was grown in LEX at 37°C to OD600 of 2.3. Methionine biosynthesis inhibition and IPTG-based induction were carried out according to the manufacturer's protocol. The temperature was reduced to 14°C and the culture was incubated for a further 18 hours before harvesting the cells.

Purification

Procedure
Column 1: Affinity purification, open Ni-NTA column Procedure: The supernatant was incubated with 6mL of 50% slurry Ni-NTA beads by rocking. After 1 hour incubation at 4ºC, the beads were washed with 50 mL of lysis buffer. The protein was eluted using ~20mL EB. Column 2: Size Exclusion, HiLoad 16/60 Superdex 75 Prep Grade Procedure: The eluent from from the NiNTA column was concentrated and loaded onto the size exclusion column in at 1 mL/min, fraction size 7mL. The fractions containing protein were identified on a SDS-PAGE gel.

Extraction

Procedure
Cells were harvested by centrifugation and pellets were stored in -80ºC. Prior to purification, the cell pellet was resuspended in lysis buffer. Cells were disrupted by sonication (10 minutes) and samples were centrifuged for 60 min at 70000 g.
Concentration:10 mg/ml.
Ligand
MassSpec:
Crystallization:25% PEG 3350, 0.1 M Tris pH 7.0, 0.2M MgCl2, 10 mM DTT by hanging-drop vapour diffusion. The drop was prepared by mixing 2.5 microL protein with 1 microL of reservoir solution. Crystals appear after a minimum period of 2 weeks.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Fischle,W., Wang,Y., Jacobs,S.A., Kim,Y., Allis,C.D., and Khorasanizadeh,S. 2003. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 17: 1870-1881
Flanagan,J.F., Mi,L.Z., Chruszcz,M., Cymborowski,M., Clines,K.L., Kim,Y., Minor,W., Rastinejad,F., and Khorasanizadeh,S. 2005. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438: 1181-1185.
Huang, J. and Berger, S.L. 2008. The emerging field of dynamic lysine methylation of non-histone proteins. Curr. Opin. Genet. Dev. 18, 152-158
Jacobs,S.A., and Khorasanizadeh,S. 2002. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 295: 2080-2083
Min,J., Zhang,Y., and Xu,R.M. 2003. Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev. 17, 1823-1828
Maurer-Stroh,S., Dickens,N.J., Hughes-Davies,L., Kouzarides,T., Eisenhaber,F., and Ponting,C.P. 2003. The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28: 69-74.
Nielsen,P.R., Nietlispach,D., Mott,H.R., Callaghan,J., Bannister,A., Kouzarides,T., Murzin,A.G., Murzina,N.V., and Laue,E.D. 2002. Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416: 103-1
Shi,Y. and Whetstine,J.R. 2007. Dynamic regulation of histone lysine methylation by demethylases. Mol. Cell 25(1), 1-14.
Taverna,S.D., Li,H., Ruthenburg,A.J., Allis,C.D., and Patel,D.J. 2007. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14: 1025-1040.
Wysocka,J., Swigut,T., Milne,T.A., Dou,Y., Zhang,X., Burlingame,A.L., Roeder,R.G., Brivanlou,A.H., and Allis,C.D. 2005. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 121: 859-872.