Structural basis for specific binding of human MPP8 chromodomain to histone H3 methylated at lysine 9

Abstract

Background: M-phase phosphoprotein 8 (MPP8) was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9.

Methodology/principal findings: Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15). The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits.

Conclusions/significance: Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

Figure 1. Overall structure of hMPP8 chromodomain.

(A) Sequence alignment of chromodomains of human MPP8, Drosophila HP1 and Drosophila Polycomb. Secondary structural elements (arrows for β strands and rectangles for α helices) are indicated, η represent 3₁₀ helix. Methylated H3K9 binding residues are marked by stars. Residues involved in dimerization of human Mpp8 and Drosophila Polycomb are marked by diamonds and triangles, respectively. The alignment was created with Espript (http://espript.ibcp.fr/Espript/Espript). (B) Cartoon representation of the crystal structure of hMPP8 chromodomain. The two subunits of the homodimer are colored in cyan and green, respectively, and the secondary structure regions in both proteins are marked. (C) Interactions between the two subunits of the hMPP8 chromodomain homodimer. The dimer interface is formed by strand β2 through an antiparallel arrangement. (D) Gel filtration of hMPP8 chromodomain (left panel). The column buffer was 20 mM Tris (pH 8.0), 400 mM NaCl. The molecular weight of hMPP8 chromodomain monomer was measured using tricine-SDS-PAGE (right panel).

Figure 2. hMPP8 chromodomain specifically recognizes di- and tri- methylated H3K9 peptides.

(A) and (B) Binding affinity of hMPP8 chromodomain to di- and tri-methylated H3K9 peptides was measured by SPR method (C) Overall structures of hMPP8 chromodomain in complex with histone H3K9me3 peptide. Cyan and green: hMPP8 chromodomain, yellow and magenta: methylated histone H3K9 peptide. (D) Monomer structure of hMPP8 chromodomain in complex with histone H3K9me3 peptides. Cyan: hMPP8 chromodomain, yellow: histone H3K9me3 peptide. (E) Interactions between hMPP8 chromodomain and H3K9me3 peptide. The chromodomain is shown in cartoon representation and colored in cyan. The H3K9me3 peptide is shown in a stick mode. (F) The aromatic cage accommodating trimethylated lysine 9.

Figure 3. Structural comparison of hMPP8, HP1 and Polycomb chromodomains.

(A) Superimposition of hMPP8 (cyan) with Drosophila HP1 (green), and hMPP8 (cyan) and Drosophila polycomb (green) chromodomain in complex with methylated histone H3K9 peptide (yellow and magenta, respectively). (B) The Drosophila polycomb chromodomain dimer. Two Drosophila polycomb chromodomain monomers are shown in a cartoon representation, colored yellow and green, respectively. Key residues involved in dimerization are shown in a stick mode. (C) Histone-histone and histone-chromodomain interactions of hMPP8 chromodomain in complex with H3K9me3 peptide in crystal lattice. Chromodomains are shown in a cartoon representation, colored yellow and green, respectively. Key residues involved in interactions are shown in a stick mode. (D) Peptide binding grooves of hMPP8, dHP1 and dPolycomb chromodomains. Chromodomain is shown in surface representation, and histone peptide is shown in a stick mode.