An Integrative Proteomic Approach Identifies Novel Cellular SMYD2 Substrates

Protein methylation is a post-translational modification with important roles in transcriptional regulation and other biological processes, but the enzyme–substrate relationship between the 68 known human protein methyltransferases and the thousands of reported methylation sites is poorly understood. Here, we propose a bioinformatic approach that integrates structural, biochemical, cellular, and proteomic data to identify novel cellular substrates of the lysine methyltransferase SMYD2. Of the 14 novel putative SMYD2 substrates identified by our approach, six were confirmed in cells by immunoprecipitation: MAPT, CCAR2, EEF2, NCOA3, STUB1, and UTP14A. Treatment with the selective SMYD2 inhibitor BAY-598 abrogated the methylation signal, indicating that methylation of these novel substrates was dependent on the catalytic activity of the enzyme. We believe that our integrative approach can be applied to other protein lysine methyltransferases, and help understand how lysine methylation participates in wider signaling processes

An Integrative Proteomic Approach Identifies Novel Cellular SMYD2 Substrates

Protein methylation is a post-translational modification with important roles in transcriptional regulation and other biological processes, but the enzyme–substrate relationship between the 68 known human protein methyltransferases and the thousands of reported methylation sites is poorly understood. Here, we propose a bioinformatic approach that integrates structural, biochemical, cellular, and proteomic data to identify novel cellular substrates of the lysine methyltransferase SMYD2. Of the 14 novel putative SMYD2 substrates identified by our approach, six were confirmed in cells by immunoprecipitation: MAPT, CCAR2, EEF2, NCOA3, STUB1, and UTP14A. Treatment with the selective SMYD2 inhibitor BAY-598 abrogated the methylation signal, indicating that methylation of these novel substrates was dependent on the catalytic activity of the enzyme. We believe that our integrative approach can be applied to other protein lysine methyltransferases, and help understand how lysine methylation participates in wider signaling processes

Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5

<div><p>Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide ‘reader’ domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.</p></div

NMR data and refinement statistics.

a<p>Values calculated for the ordered regions, as reported by PSVS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Bhattacharya1" target="_blank">[26]</a>: residues 118–183.</p>b<p>r.m.s.d calculated for residues 118–183.</p>c<p>Calculated by PSVS.</p>d<p>RPF scores <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Huang1" target="_blank">[27]</a> reflecting the goodness-of-fit of the structural ensemble to the NMR data.</p

H3K4me3 peptide titration of MLL5_PHD.

<p>(A) ¹⁵N-HSQC spectra of ¹⁵N-labelled MLL5_PHD before (black) and after (red) addition of H3K4me3 peptide. (B) Normalized chemical shift changes upon H3K4me3 binding. The normalized chemical shift perturbations for backbone ¹⁵N and ¹H_N resonances were calculated using the equation , were Δδ is the change in chemical shift in ppm.</p

Molecular surface representation of the PHD domain shown from two points of view.

<p>The surface is colored according to electrostatic potential (red for negative charges and blue for positive charges). The orientation of the domain is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone-0077020-g001" target="_blank">Figure 1</a>. MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Koradi1" target="_blank">[28]</a> was used to create this figure.</p

Surface comparison between PHD domains of PHF13_PHD, [PDB ID: 3O7A] (A, AIRE_PHD, [PDB ID 2KFT] (B) and that of MLL5_PHD (C).

<p>Peptide K4 is in orange stick, R2 in green stick. (A) PHF13's Trp255, Phe241, Met246, Thr234 are highlighted in yellow. (B) AIRE's Cys310, Asp312, Thr334, Trp335 are highlighted in magenta (C) MLL5's Trp141, Met132, His127, Thr119 are highlighted in yellow and Cys134, Asp136, Thr157, Tyr158 are highlighted in magenta. Residues for which chemical shifts have changed by more than 0.3 ppm are indicated.</p

Multiple sequence alignment of PHD domains of MLL5 in various organisms.

<p>Conserved residues are marked with red circles at the bottom <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Celniker1" target="_blank">[29]</a>. Organism of origin is shown on the left-hand-side of each sequence. Secondary structure elements of the PHD domain are shown above its sequence for clarity (α-helix as cylinder and β-strands as arrows). The residues coordinating Zn1 and Zn2 atoms are marked with blue and black dots at the top, respectively. Homologous domains are identified using protein blast against non-redundant protein database (<a href="http://blast.ncbi.nlm.nih.gov/blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/blast.cgi</a>). multiple sequence alignment is performed using clustalw2 (<a href="http://www.ebi.ac.uk/tools/clustalw2" target="_blank">www.ebi.ac.uk/tools/clustalw2</a>).</p

Comparison of PHD domains of MLL5 and PHF13 proteins.

<p>Ribbon representation of the domains with superimposed backbone, MLL5_PHD in blue and unbound PHF13_PHD (PDB ID 3O70) in orange.</p

MLL5_iso1 and histone H3 peptides complexes.

<p>Purified MLL5_iso1 incubated with biotinylated H3 peptides and complex was pulled down using streptavidin agarose beads and detected using anti-MLL5 antibody. (A) Pull down assay using H3 peptides with methylation at different lysine sites. (B) Pull down assay using H3 peptides with varying degrees of methylation at the K4 site.</p