SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

SETD2 催化组蛋白 H3 的第 36 位赖氨酸的甲基化，并且它与许多疾病有关。我们研究了 SETD2 的底物序列特异性，并确定了九种额外的肽和一种蛋白质 (FBN1) 底物。我们的数据表明，SETD2 在其特异性谱的几个位置上强烈偏爱与 H3K36 序列中的氨基酸不同的氨基酸。基于此，我们设计了一种包含四个氨基酸交换的优化超级底物，并通过 SETD2 的定量甲基化测定表明超级底物肽的甲基化效率比 H3K36 肽高约 290 倍。蛋白质甲基化研究证实了体外和细胞中超级底物的非常强的 SETD2 甲基化。我们用含有目标赖氨酸到蛋氨酸突变的结合超底物肽解决了 SETD2 的结构，这揭示了涉及三个取代残基的更好相互作用。我们的数据表明，底物序列设计可以大大提高蛋白质赖氨酸甲基转移酶的活性。