Structural Insights for MPP8 Chromodomain Interaction with Histone H3 Lysine 9: Potential Effect of Phosphorylation on Methyl-Lysine Binding

Abstract

M phase phosphoprotein 8 (MPP8) harbors a N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine 9 containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80, Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side chain hydroxyl oxygen of Tyr83 suggests the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).

A Histone Methylation Network Regulates Transgenerational Epigenetic Memory in C. elegans

“…The closest mammalian EAP-1 homologue is MPP8 (full length protein: 27.33% identity, chromodomain: 50% identity), which binds methylated H3K9 (Chang et al, 2011; Kokura et al, 2010). In vitro binding assays, using purified chromodomain (EAP-1 chromo ) or full length EAP-1 fused to GST, showed that EAP-1 selectively binds to H3K9 methylated peptides (Figures 6A and S6A).…”

“…In vitro binding assays, using purified chromodomain (EAP-1 chromo ) or full length EAP-1 fused to GST, showed that EAP-1 selectively binds to H3K9 methylated peptides (Figures 6A and S6A). Using MPP8 as a guide, we identified F24, W45 and Y48 in EAP-1 as the predicted aromatic cage forming residues (Chang et al, 2011) (Figure 6A). Mutation of each of these sites to alanine eliminated binding of EAP-1 to H3K9 methylated peptides (Figures 6A, S6B, and data not shown).…”

Application of histone modification-specific interaction domains as an alternative to antibodies

“…The K D values of HMIDs used here are in the high nanomolar to low micromolar range (MPHOSPH8 Chromo binding to H3K9me3 peptides: 120 nM [Bock et al 2011a], 140 nM [Chang et al 2011]; ATRX ADDH3K9me3: 1.4 mM [Dhayalan et al 2011]; DNMT3A PWWPH3K36me3: 64 mM ]; CBX7-H3K27me3: 22 mM [Bernstein et al 2006]). In contrast, typical antibody binding constants of commercial histone tail antibodies are in the low nanomolar to low micromolar range (for example, two papers reported K D values of 0.2, 4, 60, 83, 520, 820, 1000, 2200, and 2700 nM for several representative commercial antibodies [Nishikori et al 2012;Hattori et al 2013]).…”

Chromodomain Ligand Optimization via Target-Class Directed Combinatorial Repurposing

“…11,12,13 While the diversity of methylated marks interpreted by chromodomains is vast, many of the chromodomains bind the methylated histone consensus sequence ARKme3S. 14,15,16,17 This common recognition motif interacts with the well-conserved three-stranded anti-parallel beta sheet and C-terminal alpha helix of the chromodomains to form a beta sandwich. For some chromodomains, induced fit binding of the histone peptide results in formation of the aromatic cage that is critical for Kme recognition.…”