Human embryonic ectoderm development (EED) in complex with a trimethylated H4K20 peptide

Target ID:

EED

Deposition Date:

Friday 4th September 2009

Families:

Miscellaneous

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3JPX

Authors:

Xu C, Bian CB, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J

Site:

Toronto

ICM Datapack:

ICM Datapack

3JPX

tabs

Structure Details

EED is a WD40 repeat protein, which contains 7 WD40 repeats in its C-terminal domain (aa 81 to 441) preceded by a small N-terminal domain (aa 1-80). The seven blades are symmetrically arranged around a pseudo-symmetry axis and a narrow channel runs through the middle of the β-propeller structure. The crystal structures of apo-EED and EED-histone peptide complexes were almost identical, with an RMSD of ~0.2Å for all aligned Cα atoms. As predicted from the structural analysis, EED utilizes the smaller top surface of the β-propeller to recognize methylated histone peptides in contrast to EZH2 binding interface of EED at the bottom of WD40.

In the complex of EED-H3K9me3, the side chain of the trimethylated lysine is inserted into an aromatic cage, formed by Phe97, Tyr148 and Tyr365. Phe97, Tyr148 and Tyr365 are located on beta strands 1A, 2A and the loop between 5D and 6A, respectively. The aromatic rings of these three residues make close contact and are oriented roughly perpendicular to each other. Phe97, Tyr148 and Tyr365 form the three sides of the methyllysine-binding pocket, which establishes van der Waals and cation-π interactions with trimethylated ammonium group. The extended aliphatic chain of the methyllysine lies against the ring plane of Trp364, further enhancing the hydrophobic interaction between the protein and methylated lysine.

The trimethylysine binding mode of EED appears similar to that of other classical trimethylated histone binding proteins, such as like HP1, Polycomb, EAF3 and JMJD2A, which all have a wide hydrophobic pocket and do not have a methylation state selectivity filter such as that exhibited in MBT and 53BP1 tudor proteins. Consistent with this binding mode, the WD40 domain of EED selectively recognizes trimethylated histones, which is confirmed by our fluorescence polarization binding assays.

Materials and Methods

EED

Two fragments of human EED (amino acids 40–441 and 76–441) covering the WD40 repeats were subcloned into a modified pET28 GST-LIC vector. The recombinant proteins were overexpressed at 16 °C in Escherichia coli BL21 (DE3) Codon plus RIL (Stratagene) as N-terminal GST-tagged fusion proteins and were purified by affinity chromatographyon glutathione-sepharose (GE Healthcare).

Lysis buffer and binding buffer: 20 mM Tris, pH 7.5, 400 mM　NaCl, with or without 1 mM DTT

elution buffer: 20 mM Tris, pH 7.5, 400 mM NaCl, 15 mM Glutathione

After cleavage using thrombin (Sigma Aldrich) on the column at room temperature for 4–5 h, the flow-through was collected and purified further by size exclusion chromatography(Superdex 200; GE Healthcare). gel filtration buffer: 20 mM Tris, pH 7.5, 1 mM DTT, 200 mM NaCl.

The proteins were collected and then concentrated to 15 mg∕mL in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and 1 mM DTT.

Crystallization:Before crystallizatin, Concentrated EED WD40 was mixed with H4K20me3 peptide at ration of 1:3. Crystals of human EED WD40 domain in complex with H4K20me3 was grown at 291 K using the hanging drop method by mixing equal volumes of 0.1M BTP, 3.5M sodium formate, 10 mM TCEP chloride, and 15 mg/mL protein/ligand complex. The crystals were cryoprotected by cryoprotectant consisting of 100% reservoir solution.