Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

<div><p>Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.</p> </div

Structural superposition of <i>T. brucei</i> Hsp83 NTD complexes.

<p>(<b>^*</b>) Root mean square deviation (r.m.s.d.) values in Å for all atoms superposition (residues 1 to 206). R.m.s.d. between non-crystallographic symmetry (NCS) mates is shown in italic.</p

Protein-ligand interaction maps of <i>T. brucei</i> Hsp83 NTD complexes.

<p>The Hsp83 NTD-AMPPNP interaction map was derived from the <i>L. major</i> complex structure (PDB entry 3U67). The gray region represents the adenosine binding pocket; additional residues in common with the nucleotide analogue complex structure are highlighted in blue.</p

<i>T. brucei</i> Hsp83 NTD affinity^* and parasite growth inhibition measurements.

<p>(<b>^*</b>)Affinity measurements by ITC and DSF methods are reported.</p><p><b><i>a</i></b>) Human Hsp90α; <b><i>b</i></b>) Human Hsp90β; <b><i>c</i></b>) Human TRAP-1; <b><i>d</i></b>) Yeast Hsp83. N.D. – not determined.</p

Crystal structure of <i>T. brucei</i> Hsp83 NTD.

<p>(A) A cartoon representation of the parasitic protein, the nucleotide lid region is highlighted in magenta, α-helices 3 and 4 are shown in light green. The ATP mimetic shown to indicate the position of the nucleotide binding site was derived from the <i>L. major</i> complex structure (PBD entry 3H80). (B) NTDs structural overlay between <i>Tb</i>Hsp83 (cyan) and human αHsp90 (yellow – PDB entry 3QDD). (C) Structural overlay of <i>Tb</i>Hsp83 NTD inhibitor complexes. Two orthogonal views of ribbon representations of the three complexes and the compounds are indicated as sticks. The structures are colored as follows, compound <b>1</b> complex – cyan; thienopyrimidine derivative compound <b>3</b> – green; benzamidine derivative compound 4 – brown.</p

Hsp90 inhibitors.

<p>Chemical representation of the compounds co-crystallized with <i>T. brucei</i> Hsp83 NTD and the non- commercially available compounds used in this study. All the compounds are listed in the supplementary material (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002492#pntd.0002492.s002" target="_blank">Fig. S2</a>).</p

Thermal shift assay results and thermodynamic parameters of <i>T. brucei</i> Hsp83 NTD interactions with inhibitors.

<p>(A) The Δ<i>T</i>_m values obtained by DSF are presented in this bar representation for a subset of the Hsp90 inhibitors tested. The DSF results for all compounds tested are available as supplementary material. (B) Binding Interactions Average values for Δ<i>G</i>, Δ<i>H</i>, -<i>T</i>Δ<i>S</i> are given for protein-compound interactions measured by ITC. The Δ<i>T</i>_m values derived from DSF studies are also indicated. The raw ITC graphs are available as a supplementary material.</p

Catalytic activity and substrate/cofactor binding of EZH2 (520-746) and the trimeric (EZH2-EED-SUZ12) complex.

<p>(A) The full-length trimeric complex (●) was active, and the crystallized EZH2 construct (○) was not. Activity assay conditions were optimized for the full length EZH2 in complex with EED and SUZ12 as a control. Kinetic analysis shows that the trimeric complex binds SAM (B) and a histone peptide (C) (K_m SAM: 900 ± 100 nM; K_m peptide: 205 ± 25 nM; k_cat: 24 ± 2 h^-1). Apparent kinetic parameters are the average of three measurements ± standard deviation. ITC shows that the crystallized construct binds neither SAM (D) nor the peptide substrate (E). </p

EZH2 adopts the canonical fold of SET domain methyltransferases.

<p>(A) Linear domain architecture of EZH2 showing the crystallized construct. Residue numbers according to GenBank isoform C (Uniprot isoform 1). (B) The catalytic SET domain (yellow) is folded as previously described for other histone methyltransferases such as EHMT1/GLP and MLL, but the post-SET domain is largely unresolved and its first five residues (blue) are oriented away from its expected position. The unique CXC domain adopts a novel conformation including two clusters of three Zn ions (light blue spheres). (C) A mesh representation of the EZH2 structure in the same orientation. The cofactor is expected to bind at the junction of the SET, post-SET and I-SET (cyan) domains. (D) Residues forming the substrate lysine-binding channel in EHMT1/GLP (beige – PDB code 2RFI) are structurally conserved in EZH2 (color coding as in A-C).</p

ATPase kinetic parameters for <i>T. brucei</i> Hsp83 and reported activities for other Hsp90s^*.

<p>(^*) values ± standard deviation.</p