New publication in "Communications Biology"

New publication in "Communications Biology"

The PRDM9 protein lysine methyltransferase is essential in meiotic recombination where it trimethylates H3K4 and H3K36 in chromatin. However, it is not known how this enzyme can specifically methylate these two substrates despite their dissimilar amino acid sequences. By combining biochemical and modelling techniques, we show here that this exceptional dual substrate specificity is based on a bipartite peptide recognition cleft, comprising one peripheral binding site specific for the H3K4 peptide but tolerating H3K36, and one central binding site with the opposite preferences. The N-terminal part of the H3K4 peptide and R2 are bound by a network of PRDM9 residues in a bent conformation that sterically excludes the continuation of the peptide chain. The H3K36 peptide contains G33 and G34 in this part allowing the peptide chain to follow the binding path of the R2 side chain in the H3K4 complex. Because of this, a continuous peptide can be bound in the H3K36 binding mode. Conversely, in the central part of the substrate binding pocket, hydrophobic residues as those found in H3K36 are highly preferred, enabling an accurate readout and efficient methylation of the H3K36 sequence. Still, the residues found in H3K4 in this region are tolerated which in combination with the strong recognition of the N-terminal part of H3K4 in the peripheral region of the binding site allows to specifically methylate the H3K4 substrate as well. Based on our data, site-directed mutagenesis of residues involved in PRDM9-peptide contacts allowed us to modulate the K4/K36 preferences strongly. In our protein engineering experiments, PRDM9 mutants were identified with elevated preference for H3K4 (H3K4 >> H3K36), with lost preference (H3K4 ≈ H3K36), and even with inverted preference (H3K36 > H3K4). One additional mutant showed very low activity I339Q mutant, but in this case misfolding of the purified protein cannot be excluded. These findings document that the remarkable substrate recognition by PRDM9 with dual specificity can be tuned towards the preference for one or the other peptide.