Human fragile X mental retardation-related protein 1, Agenet domain

Target ID:

FXR1

Deposition Date:

Friday 27th November 2009

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigeneticsNeurobiology

Structure type:

apo

Download Coordinates:

3KUF

Authors:

Guo YH, Adams-Cioaba M, Bian CB, Min JR

Site:

Toronto

3KUF

tabs

Structure Details

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 [1]. 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. FXR1 (Fragile X mental retardation syndrome related protein 1) is a part of FMR1 family that counts two other members FMR1 ( Fragile X mental retardation protein 1) and FXR2 (Fragile X mental retardation syndrome related protein 2)[2-4]. The three proteins are highly homologues, particularly in their N-terminal half (with≥70% amino acid sequence identify), and share the same functional domains: the Tudor domain, the two KH domains, and the RGG box. They are thus supposed to have very similar function[5] and are ascribed to royal family proteins.

FXR1 is a 62kDa protein comprising N-terminal tandem Tudor Domains, a helix-loop-helix motif, tandem KH-type domains and an RGG-box. The crystal structure of residues 2 to 132 of the full-length FXR1 was identified to comprise a tandem Tudor domain. The N-terminal Tudor domain (Tud1) spans residues 2 to 49. This region comprises four highly twisted antiparallel β-strands that form a canonical Tudor barrel and exhibit only 0.95 Å r.m.s deviation, with 75% sequence identity, on alignment of FXR1 with FXR2. The N-terminal
Tudor (Tud1) domains also show a high degree of structural homology between the paralogues and with the SMN protein (PDB: 1g5v), PHF19 (PDB: 2e5q), and the HIV-1 integrase binding domain (PDB: 1ihv). Alignment of the Tud1 with Tud2 for each of the paralogues also reveals a high degree of structural conservation between the individual Tudor domains (1.4Å r.m.s.d.). However, while structurally the Tudors are very similar, the Tud1 domains exhibit much great sequence conservation with one another than is observed between the Tud 1 and Tud2 from a single protein. Owing to the previously published data regarding the potential interaction between FMR1 with trimethylated lysine[6], the ability of the FXR1 and FXR2 paralogues to interact with peptides derived from histone tails carrying the various post-translational modifications was assessed by fluorescence polarization. Titration of the tandem Tudor domains indicated potential interactions with H3K4me3, H3K9me3 and H4K20me3.

Materials and Methods

FXR1

PDB:3KUF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC028983
Entry Clone Source:MGC: AT42-B4
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:mhhhhhhssgrenlyfqgAELTVEVRGSNGAFYKGFIKDVHEDSLTVVFENNWQPERQVPFNEVRLPPPPDIKKEISEGDEVEVYSRANDQEPCGWWLAKVRMMKGEFYVIEYAACDATYNEIVTFERLRPVNQNKTVKKNTFFKCTVD
Vector:pET28-MHL

Growth

Medium:M9 SeMET growth 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 2mL. 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:1.4M (NH4)2SO4, 0.1 M HEPES pH 6.8, 0.25M NaCl, 10 mM DTT by sitting-drop vapour diffusion, using micro seading after equilibration for 3 hours. The drop was prepared by mixing 1 microL protein with 1 microL of reservoir solution. Protein concentration is 7.25mg/ml. Crystals appear after a period of 2 days.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using mother liquor containing 20% glycerol, and flash frozen in liquid nitrogen. Diffraction data were collected APS 19-ID to 2.75 Å.
Data Processing:

References

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.
Coy, J. F.; Sedlacek, Z.; Bachner, D.; Hameister, H.; Joos, S.; Lichter, P.; Delius, H.; Poutska, A. 1995. Highly conserved 3' UTR and expression pattern of FXR1 points to a divergent gene regulation of FXR1 and FXR2. Hum. Mol. Genet. 4:2209-2218.
Siomi, M. C.; Siomi, H.; Sauer, W. H.; Srinivasan, S.; Nussbaum, r. L.; Dreyfuss, G. 1995. FXR1, an autosomal homolog of the fragile X mental retardation gene. EMBO J. 14, 2401-2408
Zhang, Y. O'Connor, J. P.; . 1995. The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 14, 5358-5366
Imbert, G.; Feng, Y.; Nelson, D.; Warren, S.T.; Mandel, J.-L. 1998. FMR1and mutations in Fragile X syndrome: molecular biology, biochemistry, and genetics. In: Warrren, S.T.; Wells,R.D.; eds. Genetic instabilities and hereditary neurological diseases. New York: Academic Press; 27-53.
Ramos, A.; Hollingworth, D.; Adinolfi, S.; Castets, M.; Kelly, G.; Frenkiel, T.A.; Bardoni, B.; Pastore, A.; 2006.The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction. Structure. 14, 21-31