Human tudor and KH domain containing

Target ID:

TDRD2

Deposition Date:

Sunday 30th November 2008

Families:

Methyl-lysine readers

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3FDR

Authors:

Amaya MF, Adams M, Guo Y, Kozieradzki I, Edwards AM, Arrowsmith CH, Weigelt J, Bountra C, Bochkarev A, Min J.

Site:

Toronto

3FDR

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.

TDRD2 is a 67 kDa protein comprising two N-terminal hnRNP K homology (KH) domains and a single Tudor domain toward the C-terminus. The KH domain provides an important clue to in vivo function, as homologues utilize this domain for RNA binding. It has been suggested that the Tudor domain of TDRD2 may be important for interaction with the methylated arginine residues present in the tails of Sm proteins, which comprise the RNA spliceosome core (Cote and Richard, 2005). Two dominant isoforms have been identified, with isoform 2 lacking 44 C-terminal amino acids, though it is believed that several splice variants with varying distances separating the KH repeat from the Tudor domain also exist (Lamb et al 2000).

The Tudor domain of TDRD2 retains a canonical β barrel fold with a core that is highly similar to the SMN and p100 Tudor domains. Also present is an aromatic cage consisting of two phenylalanine, one tyrosine, one leucine and an aspartic acid.

Materials and Methods

TDRD2

PDB:3FDR

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_006853
Entry Clone Source:
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgrenlyfq*g. The tag was removed for crystallization.
Host:E. coli BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:gGSRSLQLDKLVNEMTQHYENSVPEDLTVHVGDIVAAPLPTNGSWYRARVLGTLENGNLDLYFVDFGDNGDCPLKDLRALRSDFLSLPFQAIECS
Vector:pET28-MHL

Growth

Medium:TB
Antibiotics:
Procedure:A fresh transformation was used to inoculate 20 mL LB media containing 50 µg/mL kanamycin. The culture was grown overnight at 37°C with shaking. The next day this starter culture was used to innoculate 2L of TB medium which contained 50 μg/mL kanamycin. The culture was grown in LEX at 37°C to OD600 of 2.3 and was induced with the addition of 0.5 mM IPTG. The temperature was reduced to 16°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: HiTrap Q HP 5mL Procedure: The eluent from the Ni column diluted 1:20 in Buffer A and manually loaded on the column. The protein was then eluted via a linear gradient from 0 - 100% of buffer B.

Column 3: Gel filtration, HiLoad 16/60 Superdex 75 Prep Grade Procedure: The eluent from from the IEX column was loaded onto the gel filtration column in GF buffer at 1 mL/min, fraction size 2mL. The fractions containing protein were identified on a SDS-PAGE gel. Final step: The Histidine tag was removed via incubated with TEV protease and the resulting sample purified using the same conditions as column 3.

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:20.25 mg/ml.
Ligand
MassSpec:
Crystallization:30% PEG 4000, 0.2 M Ammonium Acetate, 0.1 M NaCitrate, pH 5.6
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Cote, J. and Richard S. 2005. Tudor domains bind symmetrical dimethylated arginines. J. Biol. Chem. 280(31), 28476-83.
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
Lamb, F.S., Barna, T.J. Goud, C., Marenholz, I., Mischke, D., and Schutte, B.C. 2000. Complex RNA processing of TDRKH, a novel gene encoding the putative RNA-binding tudor and KH domains. Gene 246 (1-2), 209-18..
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.