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Abstract
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. Using biochemical and molecular dynamics simulation approaches, we uncover that PRDM’s unique dual substrate specificity is based on distinct interaction modes of the enzyme with both substrates. Our data show that PRDM9 interacts with the H3K4 and H3K36 peptides through a bipartite peptide binding cleft, comprising one part specific for H3K4 but tolerating H3K36, and a second part with the opposite properties. Binding of the H3K4 and H3K36 peptide substrates occurs in slightly different conformations which enables the specific recognition of both substrates. While wildtype PRDM9 showed higher activity on H3K4 peptides, site-directed mutagenesis of residues involved in PRDM9-peptide contacts allowed us to strongly modulate the K4/K36 preferences creating mutants with elevated preference for H3K4, mutants with equal methylation of both substrates and even mutants with preference for H3K36. Our data illustrate evolutionary pathways to swap the sequence specificity of PKMTs by few amino acid exchanges, a process that happened several times in the divergent evolution of PKMTs.
Highlights
The PRDM9 protein lysine methyltransferase is essential in meiotic recombination where it trimethylates H3K4 and H3K36 in chromatin
PRDM9 consists of an N-terminal KRAB domain which is important for protein-protein interaction, a nuclear localization signal, and the catalytic PR/ SET domain, which is distantly related to the catalytic SET domain found in many other human PKMTs17
The absence of methylation of the K-to-A mutant substrate peptides demonstrates that K4 and K36 are the only target sites for PRDM9 methylation in the corresponding peptides confirming the ability of PRDM9 to modify H3K4 and H3K36 targets with sequence specificity
Summary
The PRDM9 protein lysine methyltransferase is essential in meiotic recombination where it trimethylates H3K4 and H3K36 in chromatin. There is still a lack of understanding of how PRDM9 manages to productively interact with both of its substrates H3K4 and H3K36, in particular as they have very different amino acid sequences (Fig. 1a) It was the aim of this study to gain a deeper understanding of characteristic molecular interactions formed between the two substrate peptides and the catalytic site of PRDM9, which establish a dual specificity peptide recognition that is unique among PKMTs. Interestingly, PRDM9 is not closely related to other PKMTs methylating either H3K4 or H3K36. Sitedirected mutagenesis of several residues involved in PRDM9-peptide contacts allowed us to modulate the K4/K36 preferences strongly revealing mutants with elevated preference for H3K4, mutants with equal methylation of H3K4 and H3K36 and even mutants with a preference for H3K36 These data illustrate potential pathways of molecular evolution to modulate PKMT specificity by few amino acid exchanges
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