Figure 3: Structure of GLP–Dnmt3a complex.

(a) ITC measurement of binding of G9a (left) or GLP (right) SET domain to unmodified mDnmt3aK44me0 peptide in the presence of one of the reaction products S-adenosyl-L-homocysteine (to prevent the enzymatic reaction as well as the release of the substrate). (b) The substrate peptide and the methyl donor analogue Sinefungin bind in two distinctive binding sites in the opposite ends of a narrow target-lysine channel (see panel c). (c) The AdoMet analogue sinefungin (adenosyl ornithine) was used to prepare the ternary complex. This mimics the step before methyl transfer, because sinefungin also carries a formal positive charge on the ɛ-amino group (N⁺-C in the place of C-S⁺ of AdoMet). The target amino group of lysine K44 and the ɛ-amino group of sinefungin are in near linear conformation (N•••N distance of 2.7 Å and N•••N–C angle of 165°). (d) Superimposition of mDnmt3a peptide (yellow) and H3K9 peptide (grey) bound with GLP SET domain (derived from PDB 3HNA³⁵). The nitrogen atoms are in blue, the oxygen atoms in red, and the carbon atoms in yellow (mDnmt3a) or grey (H3). (e) Electrostatic interactions, hydrogen bonds (dashed lines) and van der Waals interactions define GLP–mDnmt3a peptide interactions. mDnmt3a-R43 bridges between GLP–Asp1135 and GLP–Asp1145. GLP–Asp1135 hydrogen bonds with the side-chain hydroxyl oxygen atom of mDnmt3a-T41, which in turn makes an intra-molecular interaction with mDnmt3a-R43. mDnmt3a-A42 fits into a shallow hydrophobic pocket formed by Ile1218, Phe1215, and the aliphatic portion of Lys1219 of GLP. (f) Two tyrosines, Tyr 1124 and Tyr1211 (GenBank CAH71077.1), flank the target lysine K44 of mDnmt3a. (g) The corresponding tyrosines in human G9a are Y1067 and Y1154 (NCBI NP_006700.3). Coexpression of the G9a double mutant (Y1067V/Y1154F) and hDNMT3A in 293T cells failed to methylate K47 (lane 5). The whole-cell extract was analysed by western blot using the indicated antibodies.