Figure 1.
Phylogenetic tree of human histone methyltransferases.
Phylogeny is based on a multiple sequence alignment of the methyltransferase domain including the N-SET, Pre_SET, SET, I-SET, and Post-SET motifs. Substrate selectivity was extracted from Kouzarides [1]. Enzymes with solved structure are highlighted by a frame, dotted if no peptide substrate complex is available. Structures solved in the present work are framed in red.
Figure 2.
Structures of four human H3K9 HKMTs.
(A) a ternary complex of GLP with SAH and a H3K9Me substrate peptide, (B) G9a in complex with SAH, (C) SUV39H2 in complex with SAM and (D) PRDM2, highlighting Pre-Set, SET, I-SET, Post-SET domains and the conserved presence of an N-SET domain. The co-factor is shown as yellow sticks. Residues flanking un-resolved regions are connected by dotted lines. (E) The detail of the interactions between GLP and an H3K9Me peptide.
Figure 3.
Structural determinants of G9a mono/di-methylation specificity.
A model of substrate lysine-bound G9a (Top panel) was generated from the superposition of the active sites of the GPL ternary complex and GLP. Y1067 of G9a stabilizes the di-methylamine end of the substrate lysine in an orientation where the lone-pair is not facing the co-factor, thereby disfavoring transfer of a third methyl group. The Y1067F mutant loses this restriction and can tri-methylate its substrate, as indicated in the table. Previous work had shown that the F1152Y G9a mutant can only mono-methylate H3K9 [29].
Figure 4.
The I-SET domain is relatively rigid and structurally conserved.
Structural superimposition of the ternary GLP structure with G9a or Suv39H2 in complex with co-factor and with the apo-structure of PRDM2 shows that the I-SET (cyan) conformation is conserved. The backbone atoms engaged in a double hydrogen-bond with the substrate lysine observed in all available HKMT-peptide complexes are already positioned in the absence of peptide or co-factor.
Figure 5.
Backbone and side-chain contributions to peptide binding.
A–E: Both Post-SET (blue) and I-SET (cyan) backbone atoms are engaged in a network of hydrogen-bonds with the peptide main-chain (magenta). A pair of hydrogen-bonds between backbone atoms of the I-SET and substrate lysine are conserved in all available HKMTs ternary complexes to date (dotted lines flanking red arrow). F: the substrate peptide sits in a groove formed by the I-SET (cyan) and the Post-SET (blue) domains. Peptide side-chains contributing most to the interaction are shown (magenta sticks). The guanidinium group of H3R8 (R-1) makes extensive contacts with the I-SET domain.
Figure 6.
Contribution of H3K4 to H3K9 binding.
Our structures of GLP in complex with H3K9me or H3K9me2 show that H3K4 folds on top of H3R8, making polar interactions with D1131 and D1145 of GLP.
Figure 7.
Auto-inhibitory conformation of SUV39H2.
In our structure of SUV39H2, the C-terminus of the Post-Set domain (blue) adopts a conformation that positions its K264 side-chain (blue sticks) half-way into the substrate lysine channel (gray mesh). The H3K9me peptide (magenta) from a superimposed GLP-H3K9me structure is shown as a reference. SET and Post-SET of SUV39H2 are colored green and blue respectively.
Figure 8.
Electrostatic component to H3K9 peptide binding.
While the overall electrostatic profile of available H3K9 methyltransferases structures varies, the peptide-binding groove is consistently electronegative (B–E: this work, F: N. crassa methyltransferase Dim-5), in contrast with the largely positive electrostatic potential of histone tails (A). When present, the substrate peptide is shown in magenta. Residues 264–267 of SUV39H2 were partially occupying the binding site and were removed. The Post-SET domain of PRDM2 is entirely disordered and the position of the substrate lysine binding channel is indicated with a black arrow.
Table 1.
Summary of X-ray diffraction data.