Human staphylococcal nuclease and tudor domain containing 1, extended Tudor domain, in complex with PIWIL1 peptide R4me2s

Target ID:

P100

Deposition Date:

Thursday 26th August 2010

Families:

Methyl-lysine readers

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3OMC

Authors:

Lam R, Liu K, Guo YH, Bian CB, Xu C, MacKenzie F, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J

Site:

Toronto

ICM Datapack:

ICM Datapack

3OMC

tabs

Structure Details

SND1 is one of Tudor domain, together with Chromo, MBT, PWWP and Agenet-like domains, comprise the "Royal Family" protein. The "Royal family" is largely divided into two groups: methyllysine binding group (i.e. JMJD2A, 53BP1) and methylarginine binding group (SMN, TDRD group [Tdrd1-Tdrd12]). So far, crystal structures of 53BP1 tandem Tudor domains in complex with histone H4K20me2 and a JMJD2A hybrid Tudor domain with histone H3K4me3 have provided the structural basis for Tudor domain recognition of methylated lysine residues through aromatic cages. On the contrary, much less is known about the binding mechanism of methylarginine specific Tudor domains. Recently, some studies have shown that SND1 should specifically bind against PIWI proteins, one subfamily of Argonaute family proteins, especially which contains symmetrical dimethyl arginines (sDMAs).

The structures of human SND1 in complex with PIWIL1R4me2s and PIWIL1R14me2s were solved at 1.77 Å and 1.85 Å, respectively. Both of crystal structures show that the aromatic residues F740, Y746, Y763, and Y766 form a rectangle cuboid cage with the aromatic rings of F740 and Y766 parallel to each other, and those of Y746 and Y763 approximately perpendicular to those of F740 and Y766, although the R4me2s and R14me2s peptides bound the SND1 extended Tudor domain in reverse directions. In conclusion, the intact SND1 extended Tudor domain forms a wide and negatively charged binding groove, which can accommodate distinct symmetrically dimethylated arginine peptides from PIWIL1 in different orientations.

Materials and Methods

SND1

PDB:3OMC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC017180
Entry Clone Source:AU47-C12
SGC Clone Accession:P100_4; plate JMC01I:F9
Tag:N-terminal tag: MGSSHHHHHHSSRENLYFQG
Host:E. coli BL21(DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:AKQKKEKVWAHYEEQPVEEVMPVLEEKERSASYKPVFVTEITDDLHFYVQDVETGTQLEKLMENMRNDIASHPPVEGSYAPRRGEFCIAKFVDGEWYRARVEKVESPAKIHVFYIDYGNREVLPSTRLGTLSPAFSTRVLPAQATEYAFAFIQVPQDDDARTDAVDSVVRDIQNTQCLLNVEHLSAGCPHVTLQFADSKGDVGLGLVKEGLVMVEVRKEKQFQKVITEYLNAQESAKSARLNLWRYGDFRADDADEFGYSR
Vector:pET28-MHL

Growth

Medium:TB media containing 50 µg/mL kanamycin and 30 µg/mL chloramphenicol
Antibiotics:
Procedure:A fresh transformation was used to inoculate 60 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 inoculate 2L of TB growth medium containing 50 μg/mL kanamycin and 30 μg/mL chloramphenicol. The culture was grown in LEX at 37°C to OD₆₀₀ of 2.5. The temperature was reduced to 14μC and IPTG-based induction (1mM) was carried out according to the manufacturerÂs protocol. The culture was incubated for a further 18 hours before harvesting the cells. Cells were harvested by centrifugation and pellets were stored at -80°C.

Purification

Procedure
Column 1: Affinity purification, open Ni-NTA column
Procedure: The supernatant was incubated with 6mL of 50% slurry Ni-NTA beads (buffer is changed to lysis buffer prior to use) by rocking. After 1 hour incubation at 4oC, the bead mixture was transferred to an empty column and washed with wash buffer, then using 15ml elution buffer elute. Parts of eluted sample were treated with V8 and TEV which was using for crystallization.

Column 2: Size Exclusion, HiLoad 16/60 Superdex 75 Prep Grade
Procedure: The elution from the NiNTA column and treated protein were concentrated using 15 mL concentrators with a 10K Da molecular weight cut-off (Amicon Ultra-15, Millipore). The concentrated protein was loaded onto the size exclusion column at a flow rate of 1 mL/min, and 2 mL fractions were collected. The fractions containing protein were identified on an SDS-PAGE gel. Pool the fractions together and concentrated it using the 15 mL concentrators with a 10K Da molecular weight cut-off (Amicon Ultra-15, Millipore).

Extraction

Procedure
Prior to purification, the cell pellet was resuspended in lysis buffer. Cells were disrupted by sonication (100 watts, 10 minutes total time using 10 second pulses followed by 10 second rest) on ice and samples were centrifuged for 60 min at 16000RPM.
Concentration:The final concentration was 15 mg/ml. The protein yield was approximately 40 mg per liter of bacterial culture.
Ligand
MassSpec:
Crystallization:Purified protein treated with V8 and TEV protease was mixed with PIWIL1 peptides by directly adding a three-fold or five-fold molar excess of peptide to the protein solution. The crystals of SND1 appeared in a condition containing 30% PEG 1500 at 18oC overnight by the sitting drop vapor diffusion method. Prior to the data collection, the SND1 crystals were freshly soaked in the mother liquor containing 15% glycerol and flash-frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing: