Summary

The human protein lysine methyltransferase NSD2 has very important roles in development and disease but many mechanistic features of this enzyme are unclear. We investigated the substrate sequence specificity of NSD2 and designed an NSD2 super-substrate which is methylated much faster than already known substrates. Molecular dynamics simulations demonstrated that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. Moreover, we identified ATRX and FANCM as new NSD2 substrates, strengthening the connection of NSD2 with DNA repair.

Detailed description

The human protein lysine methyltransferase NSD2 (also known as WHSC1 or MMSET) catalyzes the mono- and dimethylation of histone H3 at lysine 36 (H3K36). This modification plays crucial roles in chromatin regulation, development, DNA repair, and is implicated in oncogenesis. Despite its significance, the mechanistic understanding of NSD2's substrate recognition and its full substrate spectrum remains incomplete. The authors employed peptide SPOT arrays to systematically investigate the sequence preferences of NSD2. Using overlapping histone peptide sequences centered on H3K36, they tested the methylation activity of NSD2 in vitro. They found that residues flanking the target lysine significantly influenced methylation efficiency. Surprisingly, some amino acid substitutions—different from the wild-type H3K36 sequence—enhanced NSD2 activity. By combining four of the most favorable substitutions (A31K, K37R, H38N, R39N), the authors created a peptide variant termed ssK36 (super-substrate K36), which was methylated nearly 100-fold faster than native H3K36 at the peptide level and even more efficiently at the protein level. To explain the increased catalytic efficiency of the ssK36 peptide, the authors performed molecular dynamics simulations. These simulations revealed that binding of ssK36 to NSD2 stabilized conformations of the enzyme-peptide complex that were more conducive to methyl transfer. Specifically, ssK36 engaged in stronger and more frequent contacts within the catalytic pocket and showed better alignment with the transition state geometry typical of SN2 methylation reactions. These data support the notion that the super-substrate induces a hyperactive enzyme conformation.

Based on the established substrate motif, a proteome-wide search was conducted using nuclear-localized proteins, as NSD2 is predominantly nuclear. This search identified 226 candidate peptides. These candidates were synthesized as 15-mer peptides with the lysine of interest in the center and tested in methylation assays using radioactive AdoMet. Of these, 25 peptides showed strong methylation signals and were further validated using lysine-to-alanine mutants to confirm the methylation site. Out of these, 22 peptides were confirmed to be methylated by NSD2 at the predicted lysine residues. Finally, the authors confirmed NSD2-dependent methylation for two proteins ATRX and FANCM, specifically at lysine residues K1033 and K819 respectively. To confirm methylation in a cellular context, the authors co-expressed NSD2 along with YFP-tagged ATRX or FANCM in HEK293 cells. Immunoprecipitated substrate proteins were probed with an anti-H3K36me1 antibody. Methylation was only detected in samples co-expressing NSD2, confirming that NSD2 methylates ATRX and FANCM in cells. This establishes ATRX and FANCM as bona fide non-histone substrates of NSD2. These findings suggest broader physiological and pathological roles of NSD2, particularly in genome stability and cancer biology.